Recombinant herpes simplex gb-gd vaccine

ABSTRACT

Vaccines and therapeutic compositions and methods for their production and use against Herpes Simplex Virus (HSV) are provided employing recombinant HSV glycoproteins B and D. 
     The following E. coli HB 101 strains were deposited at the A.T.C.C., where the plasmid indicates the plasmid employed to transform the strain; pH203; pHS112; pHS114; pHS127A and pHS206 were deposited on Apr. 4, 1984, and assigned Accession Nos. 39649-39653, respectively; pYHS109 and pYHS118 were deposited on Jul. 11, 1984, and given Accession Nos. 39762 and 39763, respectively.

This application is a continuation of Ser. No. 07/079,605, filed Jul.29, 1987, now abandoned, which is a continuation-in-part application ofSer. No. 06/921,213, filed Oct. 20, 1986, now abandoned, which is acontinuation-in-part application of Ser. No. 06/597,784, filed Apr. 8,1984, now abandoned, and Ser. No. 06/631,669, filed Jun. 17, 1984, nowU.S. Pat. No. 4,618,578.

BACKGROUND OF THE INVENTION Field of the Invention

The herpes viruses include the herpes simplex viruses, comprising twoclosely related variants designated types 1 (HSV-1) and 2 (HSV-2). Thesetypes cross react strongly but can be distinguished by neutralizationtitrations. HSV-1 and HSV-2 are responsible for a variety of humandiseases, such as skin infections, genital herpes, viral encephalitisand the like.

The herpes simplex virus is a double stranded DNA virus having a genomeof about 150 to 160 kbp packaged within an icosahedral nucleocapsidenveloped in a membrane. The membrane includes a number ofvirus-specific glycoproteins, the most abundant of which are gB, gC, gDand gE, where gB and gD are cross-reactive between types 1 and 2.

It is a matter of great medical and scientific interest to provide safeand effective vaccines for humans against both HSV-1 and HSV-2 and,where infection has occurred, therapies for treatment of the disease.

One promising approach has been the use of isolated glycoproteins, whichhave been shown to provide projection when injected into micesubsequently challenged with live virus. However, the availability ofthe Herpes Simplex glycoproteins has heretofore been primarily dependentupon the growth of the virus and the isolation of the membranousproteins. The problems of commercial production of the glycoproteinsassociated with the handling of a dangerous pathogen, the maintenance ofthe virus in cell culture, the isolation of the glycoproteins free ofthe viral genome or portions thereof, have substantially precluded theuse of the glycoproteins as vaccines. It would therefore be desirable toprovide vaccines employing glycoproteins produced by methods other thanby growth of the virus and isolation of the membranes proteins.

There is also substantial interest in developing methods forprophylactically treating herpes infections. Since viral infections arenormally resistant to treatment with antibiotics, other techniques whichdo not have significant side effects are of great interest.

DESCRIPTION OF THE RELEVANT LITERATURE

Eberle and Mou, J. of Infectious Diseases (1983) 148:436-444, report therelative titers of antibodies to individual polypeptide antigens ofHSV-1 in human sera. Marsden et al., J. of Virology (1978) 28:624-642,report the location of a gene for a 117 kilodalton (kd) glycoprotein tolie within 0.35-0.40 map units on the genetic map of HSV by intertypicrecombination between HSV-1 and HSV-2. Ruyechan et al., ibid. (1979)29:677-697, also report the mapping of glycoprotein B gene to liebetween 0.30-0.42 map units. Skare and Summers, Virology (1977)76:581-595, report endonuclease cleavage sites for EcoRI, XbaI andHindIII on HSV-1 DNA. Roizman, Ann. Rev. Genetics (1979) 13:25-57.reports the organization of the HSV genomes. DeLuca et al., Virology(1982) 122:411, map several phenotypic mutants thought to lie in the gB1structural gene between 0.345 to 0.368 map units.

Subunit vaccines extracted from chick embryo cells infected with HSV-1or HSV-2 are described in U.S. Pat. Nos. 4,317,811 and 4,374,127. Seealso, Hilfenhaus et al., Develop. Biol. Standard (1982) 52:321-331,where the preparation of a subunit vaccine from a particular HSV-1strain (BW3) is described. Roizman et al., ibid, (1982) 52:287-304,describe the preparation of nonvirulent HSV-1 x HSV-2 recombinants anddeletion mutants which are shown to be effective in immunizing mice.Watson et al., Science (1982) 218:381-384, describe the cloning and lowlevel expression of the HSV-1 gD gene in E. coli, as well as expressionof a cloned fragment by injection into the nuclei of frog oocytes. Theyalso present the nucleotide sequence for the gD gene. Weis et al.,Nature (1983) 302:72-74, report higher level expression of gD in E.coli. This polypeptide elicits neutralizinp antibodies in rabbits.Berman et al., Science (1983) 222: 524-527, report the expression ofglycoprotein D in mammalian cell culture. Lasky et al., Biotechnology(June 1984) 527-532, report the use of this glycoprotein D for theimmunization of mice. Cohen et al., J. Virol. (1984) 49:102-108, reportthe localization and chemical synthesis of a particular antigenicdeterminant of gD, contained within residues 8-23 of the mature protein.

"Therapeutic" use of preparations of membrane proteins from HSV-infectedcells for post-infection vaccine in humans are reported by Dundarov, S.et al., Dev. Biol. Standard (1982) 52:351-357; and Skinner, G. R. B. etal., ibid. (1982) 52:333-34.

SUMMARY OF THE INVENTION

Novel vaccines and therapeutic compositions against Herpes Simplex VirusTypes 1 and 2 and methods for their production and use are provided.These vaccines and therapeutics employ a combination of virus specificpolypeptides produced by recombinant DNA technology. Particularly, HSVgB and gD were produced in modified mammalian and yeast hosts andemployed in combination as vaccines. They may be used for theprophylaxis and treatment of herpes simplex viral infections in animals,including humans.

Accordingly, one aspect of the invention is a vaccine which contains animmunogenically active HSV gB polypeptide in combination with animmunogenically active HSV gD polypeptide. These immunogenically activepolypeptides are prepared by expressing recombinant DNA constructs ineukaryotic host cells. The polypeptides are present in an amount whichis effective to produce an immune response in a mammal.

Another aspect of the invention is a method for preparing a vaccineagainst herpes simplex virus. This method consists of synthesizingimmunogenically active HSV gB and gD polypeptides in eukaryotic hostsvia the expression of recombinant DNA constructs, isolating thepolypeptides, and formulating them in immunogenic amounts with apharmacologically acceptable carrier.

Another aspect of the invention is a method for immunizing a mammalagainst herpes virus, wherein the mammal is vaccinated with the abovedescribed vaccine. The vaccination may occur before primary infection,in which case it acts to prevent primary infection, or after primaryinfection, in which case it acts to prevent or alleviate recurrentsymptoms of the infection.

Yet another aspect of the invention is a method for producing animmunogenically active HSV gD2 polypeptide. This method consists ofgrowing mammalian cells which have been modified to include a DNAconstruct capable of expression in mammalian cells. The DNA constructcontains an oligonucleotide encoding an immunogenically active gD2polypeptide; the oligonucleotide is flanked by transcriptional andtranslational regulatory sequences, wherein at least one of theregulatory sequences is not of HSV origin. The DNA construct is joinedto a system for its replication in the host mammalian cells. Thepolypeptide produced by the mammalian cells is harvested and isolated.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows physical maps of HSV-1 and HSV-2, an EcoRI cleavage map forthe prototype isomer arrangement, and a HindIII restriction map ofHSV-2.

FIG. 2 shows a restriction map of the region of HSV-1 map which encodesgB1.

FIG. 3 is a restriction map of gB1 coding region.

FIG. 4 shows the DNA amino acid sequences of gB1 and gB2.

FIG. 5 is a flow diagram of the construction of pSV1/dhfr, a mammalianexpression vector.

FIG. 6 is a flow diagram of the construction of plasmids pHS112 andpHS114, mammalian expression vectors for gB1.

FIG. 7 is a physical map of HSV-2, indicating coding regions for gB2.

FIG. 8 is a restriction map of gB2.

FIG. 9 is a flow chart showing construction of gD1 yeast expressionvectors and construction of plasmids pYHS109 and pYHS110 which carrysynthetic sequences for gD-A and gD-B.

FIG. 10 is a partial restriction map of the gD region which notes thelocation of all the gD sequences inserted into yeast expression vectors.

FIG. 11 is a flow chart of the construction of pYHS115 which carries aportion of the naturally occurring gene gD of HSV-1 strain Patton underthe transcriptional control of the GAPDH promoter and terminator.

FIG. 12 is a flow chart for the construction of pHS132, a mammalianexpression vector for gD1.

FIG. 13A and FIG. 13B are a physical map of HSV-2 indicating the codingregion for gD2.

FIG. 14 is a flow chart of the construction of mammalian vectors forgD2.

FIG. 15 shows the effect of vaccination with recombinant gB-gD afterprimary infection on recurrent herpetic disease.

FIG. 16 is a map showing some significant features of the plasmidpHS137.

FIG. 17A shows the effect of immunization with herpes virusglycoproteins on the rate of recurrent herpetic infections.

FIG. 17B shows the difference in weekly recurrence rates between controland immunized guinea pigs.

FIG. 18 is a graph showing the effect of the time of administration ofgBgD vaccine on the recurrence of herpetic disease.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS Vaccines

The vaccines of the invention employ recombinant HSV glycoproteins B andD of both types 1 and 2. Mature (full length) gB and gD proteins may beused as well as fragments, precursors and analogs that areimmunologically equivalent (i.e., provide protection against infection)to the mature proteins. As used in the claims, the terms "glycoprotein Bpolypeptide" and "glycoprotein D polypeptide" are intended to includesuch fragments, precursors, and analogs. The recombinant gB and gDpolypeptides are produced in eukaryotic cells preferably yeast ormammalian cells, most preferably mammalian cells. Fragments will be atleast about 15 amino acids and preferably at least about 30 amino acidsin length. The vaccines may comprise a mixture of type 1 polypeptides, amixture of type 2 polypeptides or a mixture of both type 1 and type 2polypeptides.

The mixtures of gB and gD polypeptides may be used neat, but normallywill be used in conjunction with a physiologically and pharmacologicallyacceptable medium, generally water, saline, phosphate buffered saline,sugar, etc., and may be employed with a physiologically acceptableadjuvant, e.g., aluminum hydroxide, muramyl dipeptide derivatives andthe like. As shown in Example 6.4, a variety of adjuvants may beefficacious. The choice of an adjuvant will depend at least in part onthe stability of the vaccine containing the adjuvant, the route ofadministration, the efficacy of the adjuvant for the species of theindividual being vaccinated, and, in humans whether or not the adjuvanthas been approved for human use by the Federal Drug Administration. Thevaccine may be delivered in liposomes and/or in conjunction with aimmunomodulators such as interleukin 2. The vaccines may be administeredby any convenient parenteral route, e.g., intravenously,intraarterially, subcutaneously, intradermally, intramuscularly orintraperitoneally. It may be advantageous to administer split doses ofvaccines which may be administered by the same or different routes. Thevaccines may be administered prior to, and/or subsequent to an initialinfection with herpes simplex virus.

Glycoproteins B and D may be used without modification. However, whensmaller related polypeptides are used, such as fragments or the like,and their molecular weight is less than about 5000 daltons, e.g., 1500to 5000 daltons, modification may be required to elicit the desiredimmune response. The smaller haptens should be conjugated to anappropriate immunogenic carrier such as tetanus toxoid or the like.

It is also possible to link short DNA fragments encoding the gB or gDpolypeptides to genes expressing proteins from other pathogenicorganisms or viruses. In this way, the resulting fused proteins mayprovide immunity for more than one disease.

The total amount of recombinant gB and gD polypeptides employed per dosewill usually be about 10 μg to 2 mg/kg, more usually about 50 μg to 1mg/kg and particularly about 100 to 500 μg/kg of host body weight. Theratio of gB to gD in the vaccine will usually be about 0.1:1 to 10:1,more usually about 0.5:1 to 10:1 and preferably about 0.5:1 to 5:1. Thedose may be administered repeatedly at daily to weekly intervals, andusually two to four week intervals, usually not more than about two toten times.

Recombinant Glycoprotein B

The preparation of recombinant gB polypeptides is described in detail inparent application Ser. No. 597,784, the disclosure of which isincorporated herein by reference. A brief description of the materialsand methods used to make recombinant gB polypeptides follows.

FIG. 4 in the Experimental section provides the nucleotide sequence forgB1 strain Patton, as well as the amino acid sequence coded by thenucleotide sequence. FIG. 4 also shows the substantial homology betweengB1 and gB2. The nucleotide sequence may be varied in numerous ways.Various fragments may be employed having independent functions, whichmay be joined to proteins other than the mature gB. In addition, thevarious codons may be modified so as the encode for the same aminoacids, but provide more efficient expression in accordance with thenature of the host. For example, the codons may be modifed in accordancewith the frequency of occurrence of a particular codon in one or moreproteins or groups of proteins, e.g., glycolytic proteins, whichcontribute to a high proportion of the total proteins of a particularhost, e.g., yeast. In some instances one or more codons may be modifiedto code for a different amino acid, substituting one amino acid foranother amino acid, where the effect of the change is not detrimental tothe immunogenicity of the protein or to other biological factors ofinterest. It may be desirable in some instances to add amino acids tothe N-terminus or C-terminus, where such additional amino acids mayprovide for a desired result. This can be readily achieved by providingfor additional codons at the 5'- or 3'-termini of the sequence encodingthe mature gB1 or its precursor. In addition, while the amino acidsequence of gB2 may differ from that of gB1 by as much as 20 numberpercent, other strains of HSV-1 or of HSV-2 will have gB glycoproteinsthe same as or similar to gB1 strain Patton or gB2 strain 333,respectively, usually differing by fewer than 5 number percent, moreusually differing by fewer than 2 number percent, and frequentlydiffering by fewer than 0.5 number percent amino acids from the aminoacid sequence of gB1 strain Patton or gB2 strain 333.

The gB1 sequence, particularly gB1 strain Patton, may be divided intofour domains beginning at the N-terminus of the protein: firsthydrophobic region extending from amino acid 1 to about amino acid 30: aregion of variable polarity extending from the first hydrophobic regionto about amino acid 726; a second hydrophobic region extending from saidvariable polarity region to about amino acid 795, and a second variablepolarity region extending to the C-terminus at amino acid 904.

Since gB is a membrane glycoprotein, based on analogy with otherglycoproteins, the first hydrophobic region may be considered the signalleader sequence directing secretion and/or membrane location. The firstsequence of variable polarity would then be external to the membrane andserve as the recognition sequence, to the extent that gB serves as areceptor for another protein or as an immunogen in a vaccine. The secondhydrophobic sequence may serve as a transmembrane integrator sequence(often termed the "anchor"), which can be joined to other amino acidsequences to bind them to a membrane. The second variable polarity aminoacid sequence would be expected to be in the cytoplasm and, to theextent that a receptor is external to the transmembrane integratorsequence, may serve to modulate one or more cytoplasmic processes.

The polynucleotide sequence encoding for the precursor to gB orfunctional fragments thereof may be cloned and expressed by insertingthe polynucleotide sequence into an appropriate expression vector andintroducing the resulting expression product construct into a compatiblehost. The coding fragments will be less than about 0.1 map unit, usuallyless than about 0.05 map unit where 1.0, map unit is the size of theentire HSV genome. The expression vector may be a low or high multicopyvector which exists extrachromosomally or integrated into the genome ofthe host cell and may provide for secretion or excretion of thepolypeptide of interest or retention of the polypeptide of interest inthe cytoplasm or in the membrane. A large number of expression vectorshave been published in the literature and are generally available foruse in eukaryotic hosts, including yeast, e.g., S. cerevisiae, and awide variety of immortalized mammalian cells, such as mouse cells,monkey cells, hamster cells, e.g., 3T3, Vero, Chinese Hamster Ovarycells (CHO), etc or primary cell lines. Depending upon the host, wheresecretion is desired, either the native or unnatural secretory leadersequence may be employed. The processing signals for cleavage of thesecretory leader may be the natural signals or the signals associatedwith the unnatural secretory leader or both in tandem.

In order to obtain the polynucleotide sequence encoding for gB1-Patton,the location of the gB1 coding sequences on the EcoRI restrictionfragment F was mapped. Three subfragments of the F fragment wereisolated and subcloned into pBR322 (FIG. 2). DNA fragments from thesesubclones were then used to probe Northern blots of Poly A⁺ mRNAisolated from HSV-1 infected cells. Fragments which hybridized to mRNAof the size expected for gB were presumed to lie within the gB codingregion. The direction of transcription of gB was also elicited bydetermining which strand of the DNA probes hybridized with the mRNA. Toverify the identity of the gB sequence, DNA fragments were used tohybrid-select HSV-1 mRNA, which was then translated in vitro and theresulting proteins analyzed for gB using a gB specific antibody.

The gB1 coding fragment may now be manipulated in a variety of ways,including restriction mapping and sequencing, so as to establish therestriction sites and the open reading frame regions for expression. TheDNA sequence may then be restricted to provide for a sequence encodingthe entire gB precursor or fragments thereof. These sequences may thenbe inserted into an appropriate expression vector having appropriatelypositioned transcriptional and, as appropriate, translational signals.This can be achieved by filling in overhangs and providing for blunt-endligation, by employing adapters, or the like.

It is of particular interest to introduce the gene in tandem with a genecapable of amplification. Convenient genes include the dihydrofolatereductase (dhfr) gene, which can be amplified by employing methotrexate,where the dhfr gene and flanking regions are reiterated; andmetallothioneins which can be amplified with heavy metals, e.g., copper,or the like. The expression product construct can be introduced into anappropriate host by any convenient means, including transformation,transfection, calcium phosphate precipitation, etc. The host cells maythen be stressed with the appropriate biocide at levels which select foramplification of the particular gene. The cells may then be cultured andgrown to provide efficient production of the desired polypeptide.

Following the procedure described above, the polynucleotide sequencecoding for gB2 from a HSV-2 strain 333, both precursor and mature, mayalso be isolated, cloned, and manipulated to provide a construct whichmay result in expression in one or more hosts. In view of theavailability of fragments coding for gB1-Patton, these fragments may beused as probes for either localization of gB2 encoding DNA segments tospecific HSV-2 restriction fragment clone(s) or isolation of gB2 mRNAfrom infected host cells. Conveniently, a plurality of probes may beemployed coding for different regions of the gB1 gene. One selects foreither positive DNA fragment(s) or abundant mRNA having approximatelythe right size which hybridizes to the probe(s). The mRNA may then bereverse transcribed to provide cDNA and/or may be used for hybridizationto fragments of the HSV-2 genome to confirm their gB2 encoding function.Where necessary, more than one cloned fragment comprising portions ofthe gB2 structural gene may be manipulated and joined to provide theentire coding region and flanking region(s), as appropriate. The codingregion may then be introduced into an expression vector.

Recombinant Glycoprotein D

The preparation of recombinant gD1 is described in detail in parentapplication Ser. No. 631,669, the disclosure of which is incorporatedherein by reference. A brief description of the materials and methodsused to make recombinant gD polypeptides follows. A detailed descriptionof the preparation of recombinant gD2 is presented in the Experimentalsection below.

Polypeptides which are immunologically cross-reactive with naturallyoccurring glycoprotein D are produced in eukaryotic hosts, e.g., yeastand mammalian cells, such as CHO cells by recombinant DNA methodology.Production in eukaryotes provides the advantages associated witheukaryotic hosts, e.g., post-translational modification and/orsecretion. The gD polypeptides may be produced from relatively shortsynthetic DNA fragments encoding for at least about 9 amino acids toprovide haptens useful for eliciting an immune response specific for gD.

The gD DNA fragments may be of natural or synthetic origins. The naturalgD gene of HSV-1 is located on the viral genome between the shortinternal repeat (IR_(S)) sequence and short terminal repeat (TR_(S))sequence at the 3'-end thereof. Coding for the mature protein is foundon an approximately 1.6 kbp fragment located on a 2.9 kbp SacIrestriction fragment of the genome. The entire coding region for themature protein is located within a HindIII-NruI fragment of the 2.9 kbpSacI fragment. The naturally occurring gD gene may be employed with orwithout modification. Regions of the gene may be deleted and/or joinedto other DNA fragments as desired. The gD DNA fragments may be insertedin expression vectors and expressed using similar materials andprocedures as are described above for the expression of gB DNA. Thepreparation, cloning and expression of particular fragments of thenaturally occurring gD gene are described in detail in the Experimentalsection hereinafter.

The following examples are offered by way of illustration and not by wayof limitation. In the examples: Section 1 describes general proceduresused to make the recombinant proteins; Section 2 describes thepreparation of recombinant gB1; Section 3 describes the preparation ofrecombinant gB2; Section 4 describes the preparation of recombinant gD1;Section 5 describes the preparation of recombinant gD2: and Section 6describes vaccine studies using mixtures of gB and gD polypeptides.

EXAMPLES 1. Materials and Methods

The HSV-1 strain Patton and HSV-2 strain 333 viable stocks are availablefrom Dr. Richard Hyman, Hershey Medical Center, Hershey, Pa. Theseviruses can be propagated in Vero cells available from Dr. EvelynLinnette, Viro Labs, Emeryville, Calif., or from the American TypeTissue Culture Laboratory, the propagation being performed in accordancewith standard procedures. A library of HSV-1 Patton EcoRI DNA fragments(Kudler et al., Virology (1983) 124: 86-99) cloned in the EcoRI site ofthe plasmid pACYC184 (Chang and Cohen, J. Bacteriology (1978) 134:1141)can be obtained from Dr. Hyman or be independently prepared inaccordance with conventional techniques. Two HSV-2 333 clones can alsobe obtained from Dr. Hyman, namely the HindIII fragments H and Linserted into the HindIII site of pBR322 (Sutcliffe, Nucleic AcidsResearch (1978) 5:2721).

The dhfr deficient CHO cell line was obtained from Dr. Y. W. Kan(University of California at San Francisco). This cell line wasoriginally described by Urlaub and Chasin, Proc. Natl. Acad. Sci. USA(1980) 77:4216-4220. For nonselective conditions, these cells were grownin Ham's F-12 medium (available from Gibco, cat. no. 176) supplementedwith 10% fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycinand 150 μg/ml L-proline. Selective media was DME supplemented with 10%dialyzed fetal calf serum plus penicillin, streptomycin and 150 μg/mlL-proline. For methotrexate (MTX) selection, concentrated MTX stockswere prepared from MTX obtained from Lederle and added to the above DMEselective media immediately before use.

1.1 Cloning

All DNA manipulations were done according to standard procedures. See,Maniatis et al., Molecular Cloning, CSH (1982). Restriction enzymes, T4DNA ligase, E. coli DNA polymerase I Klenow fragment, and otherbiological reagents were purchased from Bethesda Research Laboratoriesor other indicated commercial suppliers and used according to themanufacturer's directions. Double-strand DNA fragments were separated on1% agarose gels and isolated by electroelution.

1.2 Isolation of RNA, Northern blot analysis and hybrid-selectedtranslation

Total RNA was prepared from HSV-1 or HSV-2 infected Vero cells at 6 hrsafter infection with multiplicity of 10 virus per cell. Cell monolayerswere washed, incubated with extraction buffer and processed as described(Pachl et al., Cell (1983) 33:335-344). Poly A⁺ RNA was prepared bypassing 2 mg total RNA over a 3 ml column of oligo dT cellulose(obtained from Collaborative Research) in 500 mM NaCl, 10 mM Tris HCl pH7.5, 1 mM EDTA, 0.1% SDS, then washing the column in 100 mM NaCl. 10 mMTris HCl pH 7.5, 1 mM EDTA, 0.1% SDS and then eluting the poly Afraction with 10 mM Tris HCl pH 7.5. 1 mM EDTA, 0.1% SDS.

For Northern blot analysis, poly A⁺ RNA was denatured with glyoxal(McMaster et al., Proc. Natl. Acad. Sci. USA (1977) 74:4835-4838),fractionated by electrophoresis on 1% agarose gels, transferred tonitrocellulose paper (Thomas, ibid. (1980) 77:5201-5205) and hybridizedwith ³² P-labeled probes.

The details of the methods used for hybrid-selected translations havebeen described previously (pachl et al., Cell (1983) 33:335-344). DNAfilters were prepared using either 3 μg of a 3.5 kb Xho-Kpn fragmentencoding gB or 2 μg of a 3.0 kb SstI-SstI fragment encoding HSV-1 gD.The filters were incubated with 40 μg of poly A⁺ RNA from HSV-1 infectedcells. Bound RNA was eluted and translated in a reticulocyte cell-freesystem (Pachl et al., J. Virol. (1983) 45:133-139). Translation productswere analyzed on 12.5% SDS polyacrylamide gels (Laemmli, Nature (1970)227:689).

1.3 DNA transfections

Transformation of COS 7 cells (Gluzman, Cell (1981) 23:175-182) or dhfrdeficient CHO cells (Urlaub and Chasin, (1980) supra) was carried outusing the procedure of van der Eb and Graham (Methods in Enz. (1980)65:826-839), as modified by parker and Stark (J. of Virol. (.1979)31:360-369). except that carrier DNA was omitted. A calcium phosphateprecipitate of plasmid DNA was prepared by mixing an equal volume ofplasmid DNA, in 250 mM CaCl₂, with an equal volume of 2× concentratedHEPES-buffered saline (2×HBS) added dropwise (1×HBS is 0.14M NaCl, 5 mMKCl, 0.7 mM Na₂ HPO₄, 2.8 mM glucose, 10 mM HEPES pH 7.0). After about20 min incubation at room temperature, 1 ml of the calcium phosphate-DNAsuspension (containing 15 μg DNA) was added to the media of cells, grownto 50% confluency on 10 cm plates. After 6-8 hrs the DNA-containingmedia was removed and the cells were incubated with 15% glycerol-1×HBSfor 4 min. The cells were then grown in nonselective media (F12) for twodays, after which the cells were split, i.e., subcultured, intoselective media. Colonies of dhfr positive cells appeared after 10 daysand were isolated after 14 days by removing the cells of a colony from adish with a Pasteur pipette. The isolated cells were transferred tomultiwell dishes for propagation.

1.4 In vivo labelling of cells and immunoprecipitation

To label with ³⁵ S-methionine, cells were grown to confluency in 3.5 cmdishes, washed once with pBS (0.14M NaCl, 2.7 mM KCl, 15.3 mM Na₂ HPO₄)and then 0.5 ml of labeling media, DME (Dulbecco's Modified Eagle mediumfrom Gibco, cat. no. 188G) without methionine plus 1% dialyzed fetalcalf serum and 400 μCi/ml ³⁵ S-methionine (>1000 Ci/mmole) was added perdish. The cells were incubated for appropriate times at 37° C. At theend of the labeling period, the media was removed and the monolayerwashed once with PBS. For a "cold" methionine chase, the labeling mediawas replaced with DME containing 2.5 mM methionine. For immuneprecipitation, cells were lysed in 0.1 ml of lysis buffer: 20 mMTris-HCl pH 8, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P40, 0.5% sodiumdeoxycholate, bovine serum albumin, 0.1% SDS, 1.0 mMphenylmethylsulfonyl fluoride, 10 mM benzamidine 1% aprotenin obtainedfrom Sigma Chemical Company. The cell lysate was scraped into tubes,briefly vortexed, and then held at 4° C. for 5-10 min. Cell debris wasremoved by centrifugation and the clarified lysate stored at -70° C.

For immunoprecipitations, cell lystates, 0.1 ml, were precleared byincubation with normal serum for 30 min at 4° C. then 50 μl of a 20%solution of protein A Sepharose (PAS) (in lysis buffer) was added andincubation continued for 30 min at 4° C. with gentle rocking. The PASwas removed by centrifugation for 1 min at 14,000× g and 5 μl of HSV-1polyclonal antibody (obtained from DAKO) or a gB-specific monoclonalantibody F3AB (obtained from Dr. John Oakes, University of SouthAlabama) was added. When the F3AB antibody was used, 0.1% SDS wasomitted from the lysis buffer. After 30 min at 4° C., 75 μl of PAS wasadded and above. PAS-immune complexes were collected by centrifugation,washed 3× with lysis buffer lacking BSA and protease inhibitors and oncewith 0.12M Tris HCl pH 7.0. Immune precipitated proteins were releasedfrom PAS by boiling in SDS sample buffer, followed by analysis on 12%polyacrylamide gels. For immune precipitation of labeled proteins fromcell media, the media was first clarified by centrifugation and then1/10 volume of 10× lysis buffer was added and proteins were precipitatedas described above.

1.5 Immunofluorescence

To analyze expression of gB or gD in COS cells or CHO clones, cells,grown in slide wells, were washed 3× with PBS, fixed with 100% methanolat -20° C. for 10 min followed by 3 more PBS washes and one wash withPBS plus 5% goat serum (GS). The fixed cells Were then incubated withthe primary antibody (HSV-1 or HSV-2 polyclonal diluted 1/100 in PBS-5%GS) for 30 min at 37° C. The cells were then washed 3× in PBS-5% GS andthen incubated at 37° C. for 30 min with the second antibody.FITC-conjugated goat anti-rabbit IgG (Cappel). diluted 1/10 in PBS-5%GS. After 4 washes in PBS-5% GS, the slides were mounted with coverslipsusing 50% glycerol--100 mM Tris HCl, pH 8 and observed in a Leitzmicroscope equipped with epifluorescent optics. Live cellimmunofluorescence was carried out as described above except that thecells were initially washed once in PBS-5% GS directly followed byincubation with the first antibody. Before mounting with coverslips, thelive cells were fixed with 5% formaldehyde in PBS. The fluoresceinstained cells were photographed using a Kodak Ektachrome film (ASA 400).

1.6 ELISA Assay

The concentration of gB protein in CHO cell conditioned medium wasmeasured by an indirect enzyme-linked immunosorbent assay (ELISA) usinga preparation of purified recombinant gB as a standard. Aliquots of 50μl of F3AB antibody diluted 1:1000 in PBS were adsorbed to the wells ofa 96-well polyvinyl chloride plate (Dynatech Laboratories, Inc.) byincubation for 1 hr at room temperature. Excess antibody was removed by3 washes with PBS-5% GS, 50 μl aliquots of media samples or the gBprotein standard diluted in PBS+1% GS were added to the wells andincubated for 1 hr at room temperature. The plates were then washed 3times with PBS+1% GS and followed by a third 1 hr incubation with 50 μlof rabbit anti-HSV-1 polyclonal antibody (obtained from DAKO) diluted1:100 in the same buffer. Excess secondary antibody was removed by 3washes with PBS+1% GS. Finally. 50 μl of goat anti-rabbit horseradishperoxidase-conjugated antibody (Boehringer Mannheim) diluted 1:500 inPBS+1% GS was added to each well for a 1 hr incubation. The wells werethen washed once with PBS+1% GS, followed by 8 washes with PBS and thendeveloped with 50 μl of 2,2'-azido-di[3-ethylbenz-thioazoline sulfonate](Boehringer Mannheim) at a concentration of 1 mg/ml in 0.1M citric acid,pH 4.0, 0.003% H₂ O₂. The color reaction was stopped after 5 minutes bythe addition of 50 μl of 10% SDS and the absorbance was read at 414 nmin a microtiter plate reader.

The concentration of gD protein was measured in similar fashion exceptthat purified recombinant gD was used as a standard, and 8D2, agD-specific monoclonal antibody (Rector et al., Infect. and Immun.(1982) 38:168-174) replaced F3AB.

1.7 Yeast transformation

Yeast were transformed (Hinnen et al., Proc. Natl. Acad. Sci. 75:1929,1978) and grown using a variety of media including selective medium(yeast nitrogen base without leucine); YEPD medium, containing 1% (w/v)yeast extract, 2% (w/v) peptone and 2% (w/v) glucose, and others asappropriate and/or detailed below. Plating medium contained 2% (w/v)agar and transformation medium 3% top agar.

2. Glycoprotein B1 2.1 Isolation, cloning and characterization of thegB1 gene

To isolate the gene for the glycoprotein gB1, DNA fragments spanning mapcoordinates 0.345 to 0.40 within the EcoRI F restriction fragment of theHSV-1 strain Patton (Skare and Summers, Virology (1977) . 76:581-595)were subcloned in the plasmid pBR322. These fragments were prepared fromthe appropriate restriction digests of the EcoRI region in the plasmidpACYC184, separated by electrophoresis on a 1% agarose gel in TAE buffer(0.04M Tris-acetate, 0.002M EDTA) and electroeluted. The isolatedfragments were ligated into pBR322 which had also been previously cutwith the appropriate restriction enzyme and treated with alkalinephosphatase. A restriction map for the entire HSV-1 genome is shown inFIG. 1, and a more detailed map of the region which was subcloned isshown in FIG. 2. Referring to FIG. 1, the conventional map is shown inthe first two lines (Roizman, 1979). The dotted line indicates the L-Sjunction. The restriction enzyme cleavage map for EcoRI for theprototype isomer arrangement is shown in the third line (Skare andSummers, 1977; Roizman, 1979) with the EcoRI fragment F denoted by thecross-hatched box. For HSV-2, the HindIII restriction map is shown inline 4 (Roizman, 1979) with the HindIII fragment H cross-hatched. Onemap unit corresponds to approximately 98.9 megadaltons or 148.9 kbp ofDNA for HSV-1 and 105.6 megadaltons or 160.5 kbp of DNA for HSV-2.

Referring to FIG. 2, the restriction enzyme sites shown in the detailedmap line (I) are E, EcoRI; B, BamHI; S, SalI; p, PstI, X, XhoI fromDeLucca et al., 1983; N, NdeI; Xn, XmnI; V. EcoRV. The BstEII sitemapped by DeLucca et al. at 0.355 is missing in this strain and there isa new PstI site at 0.357. Line II shows three plasmid subclones whichencompass the gB1 coding region. They are pHS106, which extends from theBamHI site at 0.345 to the SalI site at 0.360; pHS107 which extends fromthe SalI site at 0.36 to the SalI site at 0.388; and pHS108 which is aBamHI fragment extending from 0.345 to 0.40 map units. Line IIIindicates three probes used for mRNA mapping of gB1; line IV indicatesthe fragment used for hybrid selection; and line V shows those probesused to locate the gB2 gene (see below). The additional restrictionsites used to generate these fragments are Nc, NcoI; K, KpnI; and A,AluI.

To locate the gB1 coding region within the EcoRI F fragment, Northernblots of poly A⁺ mRNA isolated from HSV-1 infected Vero cells wereprobed with the DNA fragments indicated on the detailed map isolatedfrom plasmids pHS106 and pHS107. When HSV-1 mRNA was probed with a 0.56kb PstI-SalI fragment isolated from pHS106, a 3 kb mRNA was the majorspecies detected. When the same blot was probed with a 0.49 kb NcoIfragment, which maps about 1 kb upstream from the PstI-SalI fragment,hybridization to a 3 kb mRNA, the presumptive gB1 mRNA, was alsodetected. This suggests that the gB1 coding sequences extend at least 1kb to the left of the PstI-SalI fragment. The 3 kb mRNA does not extendbeyond the first XhoI site downstream from the PstI-SalI fragment, sincethe 0.5 kb KhoI-XhoI fragment does not hybridize to this mRNA. Thedirection of transcription of the gB1 transcription unit is right toleft (3'← 5') as evidenced by hybridization of only the 5'→3' orientedstrands of the PstI-SalI and NcoI-NcoI fragments (cloned in M13) to the3 kb gB1 mRNA.

Hybrid selected translation was performed by hybridizing HSV-1 poly A⁺mRNA with a 3.2 kb KpnI-XhoI fragment, which encompasses the regionindicated as encoding gB1. When the bound mRNA was eluted and translatedin vitro, a 100 kd protein, similar in size to gB1 from HSV-1 infectedVero cells, was detected. Confirmation of the identity of the 100 kdprotein was achieved by immunoprecipitation with a gB1-specificmonoclonal antibody. Several other proteins were also detected by hybridselection using the KpnI-XhoI fragment, probably the result ofnon-specific hybridization of mRNAs due to the high G+C content of theDNA. A similar pattern of proteins was seen when the same RNA wasselected with a 3.0 kb SstI-SstI DNA fragment encoding HSV-1glycoprotein gD, except that the 100 kd gB protein was not detected.This result indicates that gB is specific to the XhoI-KpnI fragment.

FIG. 3 is a restriction map of a 3.95 kb DNA fragment, which extendsfrom a BamHI restriction site at 0.345 to an XhoI site at 0.373 mapunits. The open reading frame for gB1 is indicated by the box and thedirection of transcription is from right to left as shown. The actualcoding region covers map units 0.348 to 0.367. The DNA sequence from theBamHI site to a non-unique AluI site at nucleotide number 3640 is shownwith the AluI site indicated by the (A). The restriction sites showninclude B, BamHI; B1, BalI; Bs, BstEII; K, KonI; Nc, NcoI; p, PstI; Pv,PvuII; S, SalI; Sc, SacI, X, XhoI; Xm, Xma3. Restriction sites are notshown for the right-hand end from the AluI site to the terminal XhoIsite. Potential glycosylation sites and hydrophobic anchor and signalregions (solid box) in the product gB1 protein are noted.

The DNA sequence was determined from the BamHI site to a non-unique AluIsite at nucleotide residue number 3640 using the M13 dideoxynucleotidesynthesis method of Sanger. Both DNA strands across the coding regionwere sequenced. The entire DNA sequence was compiled from overlappingrestriction fragments such that the sequence was read across allrestriction fragment joints. FIG. 4 shows the DNA sequence for gB1 (line3); the predicted amino acid sequence for gB1 is shown below the DNAsequence (line 4).

It should be noted that the amino acid sequence and DNA sequence for gB1presented in FIG. 4 differs from that originally presented in Table 1 ofthe parent application, Ser. No. 597,784, filed Apr. 6, 1984, thedisclosure of which is hereby incorporated by reference. The DNAsequence in said Table 1 contains an error in that an additionalnucleotide (G) is listed at position 607; this nucleotide has beendeleted in FIG. 4, which presents the corrected DNA sequence. The aminoacid sequence in said Table 1 was deduced from the incorrect DNAsequence presented therein; the sequence as presented in said Table 1 isincorrect because of the shift in reading frame due to the additionalnucleotide. FIG. 4 presents the amino acid sequence based upon thecorrected DNA sequence; the amino acid sequence in FIG. 4 has beenconfirmed by amino acid sequencing of the N-terminal region of gB1. Thischange in the deduced amino acid sequence also results in correctionconcerning the deduced position of the hydrophobic and hydrophilicregions, and the glycosylation sites in the gB1 molecule. The deductionsbased upon the corrected sequence are presented below.

Primer extension, using a 22 bp oligonucleotide (residues 473-494)indicated that the 5'-end of gB1 mRNA was located at residue 188. TheCAT and TATA transcriptional regulatory signals are presumptively atresidues 55-62 and 125-131. Starting at the ATG at residues 438-440,there is an open reading frame of 2712 nucleotides which terminates at aTGA stop codon. Two presumptive polyadenylation signals are located in a3'-non-coding region at residues 3166-3173 and 3409-3416.

The observed amino acid sequence is characteristic of a membraneprotein. There is a very hydrophobic region near the carboxy terminusstretching from amino acid residue number 726 to 795, a 69-amino acidsequence which may span the membrane. At the N-terminus the first 30amino acids are primarily hydrophobic. This hydrophobic amino aciddomain precedes a region with a high concentration of charged orhydrophilic amino acids. The hydrophobic sequence at the N-terminus mayserve as a secretory leader or signal sequence followed by processingsignals for cleavage and removal of the secretory leader. Thehydrophobic region near the C-terminus can serve as a transmembraneintegration sequence for binding the protein to the cell membrane.

The sequence data is also suggestive that there are nine possibleN-linked glycosylation sites as defined by the sequence asn-X-thr/ser(see also FIG. 3) within the hydrophilic, external domain. If the first30 amino acids are removed by processing and each of the potentialN-linked glycosylation sites are utilized with the addition of anaverage 2 kd of carbohydrate per site, the molecular weight of themature protein would be approximately 123 Kd.

2.2 Expression of gB1 in mammalian cells

Employing the above DNA sequence or fragment thereof, expression wasachieved as follows. The vector employed is a mammalian expressionvector, referred to as pSV1/dhfr. This 5.63 kb plasmid contains 2.8 kbof E. coli plasmid pBR328 sequences, including the ampicillin-resistanceβ-lactamase gen and the origin of replication. The vector also containsa selectable mammalian cell marker, the mouse dihydrofolate reductasecDNA gene (dhfr) (Nunberg et al., Cell (1980) 19:355) linked to the SV40early promoter, which directs the transcription of dhfr. Additional SV40sequences, including t antigen splice donor and splice acceptor sitesand the polyadenylation sites for early transcripts, are includeddownstream from the dhfr gene within a 1.65 kb BolII-EcoRI fragment.

The plasmid pSV1/dhfr was constructed by first isolating the 0.4 kbBamHI-HindIII fragment encoding the SV40 origin and early promoter fromplasmid pSVgt1. This SV40 fragment was then inserted into plasmid pBR328by substituting this fragment for the small HindIII-BamHI fragment ofpBR328. The dhfr cDNA gene and the SV40 splice sites and poly A sites ofpSV1/dhfr were derived from plasmid pSV2/dhfr (Mulligan and Berg, Mol.Cell Biol. (1981) 1:854-864). The 2.4 kb HindIII-RI fragment encodingthe dhfr-SV40 sequences was excised from pSV2/dhfr and inserted into theabove pBR328 plasmid by substitution for the small HindIII-RI fragmentof pBR328. The details of these constructions are given in FIG. 5.

To obtain expression of gB1, two pSV1/dhfr-gB plasmids, pHS112 andpHS114 were constructed (FIG. 6). pSV1/dhfr was restricted with BolI andHindIII excising the dhfr cDNA fragment. The resulting fragments arethen blunt-ended by filling in the overhangs With the Klenow fragment ofDNA polymerase I. A XhoI-KpnI HSV-I fragment containing the gB gene isisolated from pHS108. A portion is taken and partially digested withPvuII to generate a DNA sequence lacking the 3'-anchor region. Theresulting truncated gB1 gene lacks 580 bp from the 3'-end of the gene.Both fragments are then blunt-ended with the Klenow fragment of pol I.

Each gB blunt-ended fragment is ligated into the BglII-HindIIIrestricted pSV1/dhfr vector to provide two sets each of constructs, withthe gB1 gene in opposite orientations. The orientations having the Xhogenerated terminus proximal to the SV40 promoter, with the direction oftranscription being from the SV40 promoter to the SV40 splice sitesselected and designated pHS111 and pHS113 for the complete and truncatedgB1 genes, respectively. The two plasmids are then completely digestedwith EcoRI and partially digested with BamHI to provide a cassette whichincludes the SV40 promoter, the gB gene and the SV40 splice andpolyadenylation sites. These fragments are blunt-ended and ligated intoEcoRI digested pSV1/dhfr vectors, so as to have the gB1 gene downstreamfrom the dhfr gene and in the same orientation. The complete gB1 geneand trunctated gB1 plasmid constructs are designated pHS112 and pHS114,respectively.

The plasmids are then transfected into CHO cells deficient in dhfr usingthe calcium phosphate precipitation method as described in Materials andMethods. Transfected cells are selected by employing a selective mediumlacking thymidine, purines and glycine. Cells were isolated by removalwith a Pasteur pipette and propagated in multiwell plates. A number ofclones were isolated which were shown to produce gB byimmunofluorescence and radioimmunoprecipitation employing an HSV-1polyclonal antibody or a monoclonal antibody specific for gB. Three cellclones, pHS112-1, pHS112-9 and pHS112-23, were isolated which synthesizean intracellular form of the complete gB protein. The gB made in thesecells appears to be glycosylated, since higher molecular weight formscan be detected after a one hour pulse, followed by a 5 hr chase, ascompared to nonchased cells and about 10% of the gB is secreted into themedia. Five cell clones (pHS114-5, pHS114-6, pHS114-7, pHS114-11 andpHS114-12) expressing the truncated gB were also analyzed and shown toalso secrete some gB into the media. One of these cell lines, pHS114-7,Was chosen for further amplification with MTX. Clones were initiallyselected at 0.01, 0.05, 0.10 and 0.3 μM MTX. Three clones levels of gB,as detected by immunofluorescence, were isolated from the 0.3 μM MTXselections. By radioimmune precipitation, these clones, pHS114-0.3 μM-6,23 and 25, synthesize 2-3 times more gB during a 1 hr labeling with ³⁵S-methionine than the unamplified clone, pHS114-7. pulse chaseexperiments indicate that at least 8% of the gB synthesized in theseclones during a 1 hr pulse is secreted extracellularly by 5 hr.

Expression was also achieved using the expression vector pHS137, a mapof which is presented in FIG. 16. Plasmid pHS137 encodes a truncated gB1protein which is 690 amino acids in length after cleavage of the signalsequence.

pHS137 was constructed by digestion of pHS108 (described in Section 2.1)with XhoI and BamHI, followed by isolation of a resulting 3.5 kbfragment. The ends of this fragment were repaired to blunt with Klenow.

The blunted XhoI-BamHI fragment was partially digested with PVUII, andDNA which migrated in gels as a 2098 bp band was iolated from thepartial digest. The isolated XhoI-PVUII band was ligated into pSV7dwhich had been previously digested with SmaI, and the resulting DNA wasused to transform E. coli. The resulting bacterial clones were screenedfor a plasmid with the proper orientation of the gB1 insert.

To obtain expression, pHS137 is cotransfected with the plasmid pADdhfrinto dhfr deficient CHO cells. The resulting clones produce and secretegB1. One such clone, pHS137-7-B-50 produces 6.91±1.53 μg/ml gB1 proteinper 1-3×10⁷ cells in 24 hours in a T75 culture flask containing 10 ml ofcomplete medium.

2. 3 Expression of gB1 in yeast

Yeast expression was developed as follows. A cassette was preparedemploying the glyceraldehyde-3-phosphate-dehydrogenase (GAPD4) promoterregion and terminator region. A yeast gene library was prepared byinserting fragments obtained after partial digestion of total yeast DNAwith restriction endonuclease Sau3A in lambda-phage Charon 28 (Blattneret al., Science (1977) 196:161-169). The phage library was screened withDNA complementary to the yeast GAPDH mRNA and the yeast GAPDH gene fromone of these clones was subcloned as a 3.5 kb BamHI fragment in theBamHI site of pBR322 (pGAP-2). The GAPDH promoting-active fragments wereisolated from these clones. A HhaI-HindIII fragment of about 350 bpcontaining the 3' portion of the promoter was obtained by: a) digestionof pGAP-2 with HinfI to generate an approximately 500 bp segment whichincludes the 3' part of the promoter and a region encoding theN-terminal amino acids of GAPDH; b) resection with Ba131 to yield a 400bp fragment lacking the GAPDH coding region (3'-terminus 1 base upstreamfrom the ATG initiator codon); c) addition of HindIII linkers; and d)cleavage with HhaI. A HindIII-HhaI fragment of about 700 bp containingthe 5' portion of the promoter was ligated to the 350 bp HhaI-MindIIIfragment and treated with HindlII. The resulting 1061 bp HindIIIfragment was isolated by gel electrophoresis and cloned in pBR322(pGAP347). The GAPDH promoter fragment in pGAP347 was isolated bycleavage with BamHI (within the 5' pBR322 flanking region) and partiallywith HindIII (at the 3' end of the promoter fragment) to provide a 1407bp fragment containing a 1061 bp region of the GAPDH promoter region and346 bp of pBR322. This procedure utilized digestion of 50 μg of thepGAP347 with 10 units each of BamHI and HindIII with the resultingfragment purified by preparative gel electrophoresis in 1% agarose.

A synthetic HindIII-XhoII adapter molecule containing the codon for theinitiator met and a NcoI site for analysis was synthesized and had thefollowing sequence: ##STR1##

A third fragment was a XhoII-SacII fragment of 1187 bp containing thegB1 coding region.

A fourth fragment containing the GAPDH terminator fragment(approximately 900 bp) was isolated by SalI-BamHI digestion of a clonedfragment of the GAPDH gene with its 3' flanking region including theGAPDH termination region, so that a portion of the coding region isincluded with the termination region. The two fragments can be ligatedtogether by means of a SacII-SalI adapter: ##STR2##

These five fragments together with the cloning vector were ligated asfollows: First, the XhoII-SacII fragment (2 picomoles) was ligated to100 picomoles of each of the two adapters (HindIII-XhoII, SacII-SalI)using T4 DNA ligase. The product was isolated by preparative gelelectrophoresis in 1% agarose, providing a HindIII-SalI fragment. TheHindIII-SalI fragment (0.25 picomoles) was ligated in a single step tothe 1,407 bp BamHI-HindIII GAPCH promoter fragment (0.1 picomoles). the900 bp SalI-BamHI terminator (0.1 picomoles) and 0.02 picomoles ofBamHI-digested, phosphatased pBR322 in the presence of T4 DNA ligase.

The above reaction product was used to transform E. coli HB101. plasmidscontaining the cassette clones in pBR322 were isolated and the correctnucleotide sequence confirmed by DNA sequencing. This plasmid was thendigested with BamHI and the BamHI cassette fragment containing the gB1segment and GAPDH regulatory regions gel isolated and inserted intoBamHI-digested, phosphatased pCl/l. Plasmid pCl/l is a derivative ofpJDB219 (Beggs, Nature (1978) 275:104) in which the region correspondingto bacterial plasmid pMB9 in pJDB219 is replaced by pBR322 in pCl/l. ThepCl/l plasmid containing the 1187 bp gB1 insert and GAPDH promoter andterminator regions was designated pHS127A. This plasmid was then used totransform the yeast strain S. cerevisiae AB103.1 (α, pep 4-3, leu 2-3,leu 2-112, ura 3-52, his 4-580). Transformants were initially grown in1.0 ml of leu medium and then 50 ml of YEPD inoculated with 0.4 ml andgrown further to an absorbance of 1-3 at 650 nm (12 hr). The yeast cellswere pelleted by centrifugation at 2 krpm for 10 min at 4° C. andresuspended in 50 mM Tris-HCl, pH 8, 150 mM NaCl, 0.2% Triton X-100, 1mM EDTA and freshly added 1.0 mM phenylmethylsulfonyl fluoride and 0.1μg/ml pepstatin. The cells were repelleted and then resuspended in avolume equal to the packed cell volume in the same buffer. An equalvolume of acid-washed glass beads (diameter 0.45-0.5 mm) was added andthe yeast cells disrupted by vortexing at 4° C. for 10 min total, using1 min intervals.

The tubes were centrifuged for 15 min at 14000× g at 4° C. and thesupernatant isolated and analyzed on 10% SDS polyacrylamide gel andblotted onto nitrocellulose paper for Western analysis (Burnett, Anal.Biochemistry ](1981) 112:195). A polyclonal antibody (DAKO) to HSV-1 wasemployed as the primary antibody. Expression of an HSV specific proteinwas observed at about 44 kd, the size expected for the gB fragment.

3. Glycoprotein B2 3.1 Isolation, cloning and characterization of thegB2 gene

The gene encoding HSV-2 gB had been shown to the colinear with thecorresponding HSV-1 gB gene by analysis of HSV-1×HSV-2 intertypicrecombinants and to lie approximately between prototypic map coordinates0.30 and 0.42 (Ruyechan et al., J. Virol. (1979) 29:677-697). Thus, theHindIII H fragment of HSV-2 which spans map coordinates 0.28 to 0.40(FIG. 7) includes the gB2 coding region. FIG. 7A shows a conventionalprototype HSV-2 configuration in the first two lines. The restrictionmap for HindIII is shown in the third line. In addition to theircolinear map location, serological and heteroduplex analyses indicatethe close similarity of gB1 and gB2. Therefore, to locate the gB2 codingregion more precisely, the fragments of the gB1 gene indicated in FIG.7b, line I were used to probe Southern blots of restriction digests ofthe HSV-2 HindIII H fragment. The 0.55 kb PstI-SalI fragment thatencodes amino acids 323-506 of gB1 hybridizes to a 2.6 kb XhoI fragment,a 2.0 kb SstI fragment, and additional specific restriction fragments.The adjacent 0.49 kb NcoI fragment of gB1 hybridizes to the flanking 3.2kb XhoI band as well as a 4.2 kb SphI band. Two of these overlappingrestriction fragments were subcloned into a pBR322 plasmid derivative togenerate the plasmids pHS203 (containing the 5' end of the gene on a 2.6kb XhoI fragment) and pHS207 (containing the 3' end of the gene on a 4.2kb SphI fragment) with the map locations of the inserts shown in FIG.7B, line III.

The exact location and the identity of the gB2 gene were verified byprobing a Northern blot of poly A⁺ mRNA isolated from HSV-2-infectedVero cells with the restriction fragments of pHS203, shown in FIG. 7Bline II. Both an 0.89 kb XhoI-SmaI and a 1.29 kb Sma-Nru fragmenthybridized to an abundant 3.0 kb message, an appropriate size andrepresentational frequency for gB2 based on analogy to the analysis ofgB1 transcripts. However, the same message did not hybridize to therightmost 0.47 kb NruI-XhoI fragment. As expected a similarhybridization pattern was observed when poly A⁺ RNA prepared from HSV-1infected Vero cells was probed with these same fragments, although thesignal intensity was diminished due to the inefficiency ofcross-hybridization. Since this analysis indicated both the limit of theright hand end of the gB2 gene as well as its size, it was apparent thatthe gB2 coding sequences must extend an additional 1 kb to the left ofthose sequences contained within the 1.98 kb NruI-SphI fragment ofpHS203 into the overlapping pHS207 plasmid. Therefore, the gB2 gene wascloned as one continuous fragment of pHS203 to the 1.48 kb SohI-BamHIfragment of pHS207 and insertion into NruI and BamHI-digested pBR322 togenerate pHS208.

The HSV-2 fragment encoding gB was sequenced on both strands in itsentirety.

The nucleotide sequence for gB2 is shown in FIG. 4, line 2. Thepredicted amino acid sequence of gB2 is shown above the DNA sequence.For comparison, the DNA sequence and the amino acid sequence of gB1 fromHSV-1 strain Patton is shown below. Spaces have been inserted into thesequence to permit maximal alignment of the two proteins. All numbers ofFIG. 4 refer to the gB2 sequence. Characteristic TATA and CATtranscriptional regulatory sequences are most likely located 5' to thestart of this sequence analysis analogous to the gB1 sequence. In the 3'noncoding region, a polyadenylation signal, AATAAAAA (proudfoot andBrownlee, Nature (1976) 263:211) at residues 2744 to 2751 is theprobable termination site of the gB2 mRNA.

There is a potential transmembrane anchor region of 54 amino acids fromAla₇₄₅ to Leu798. Chou and Fasman analysis (Adv. Protein Chem. (1978)47:45-148) indicates a mixed β-sheet and α-helix potential for theentire region. However, in order to avoid orientation of polarsidechains toward the lipophilic environment of the membrane bilayer, itis likely that this region adopts an α-helical conformation (Engelmanand Steitz, Cell (1981) 23:411-422). An α-helix of this length (8.1 nm)would be more than sufficient to span a biological membrane 3 nm inthickness 2 times, placing the C-terminal domain of the protein on theexterior of the cell. Alternatively, the transmembrane domain maytraverse the membrane 3 times and include the amphipatic domainbeginning at Asp₇₂₃ that contains 4 additional charged residues. In thisanalysis, tight-packing of the 3 α-helices allows interchain hydrogenbonding between the charged residues, all of which are predicted to lieon the same face of the helix. Thus, the charged residues would bethermodynamically allowed with the membrane, as they would not interactwith the hydrophobic lipid environment. This model would localize theC-terminus of the protein within the cytoplasm. While it is notpresently possible to distinguish between the possibilities that the gBanchor spans the membrane two or three times, it is an importantconsideration in terms of positioning the C-terminus on theextracellular or cytoplasmic side of the membrane.

The C-terminal region of gB2 extends from the end of the membrane anchorregion at Leu₇₉₈ to the end of the protein at Leu₉₀₄ and contains a highdensity of charged residues. No potential N-linked glycosylation sitesare present in this portion of the type 2 protein.

The predicted gB2 protein is 904 amino acids in length and containselements characteristic of a membrane glycoprotein. After cleavage ofthe predicted 22 amino acid signal sequence, the mature, nonglycosylatedprotein would have a molecular weight of 98,221. The amino terminal 22residues contain a core of hydrophobic residues (Leu₆ to Ala₂₀) precededby a charged basic residue (Arg at position 2) and an alanine-richsignal peptidase recognition sequence, Ala₂₀ -Ser₂₁ -Ala₂₂, conformingto rules identified for preferred signal peptidase cleavage sites andthe general characteristics of eukaryotic signal peptides (Watson, Nucl.Acid Res. (1984) 12:5145-5164). Protein sequence analysis of theN-terminus of recombinant HSV-1 glycoprotein B identified the firstamino acid of the mature type 1 protein as Ala followed by Pro₃₁ Ser₃₂Ser₃₃ Pro34. Due to the conservation of the 6 amino acids centeredaround the signal cleavage recognition sequence, we assign Ala₂₃ of gB2as the first amino acid of the mature glycoprotein.

The external hydrophilic region of the protein from Ala₂₃ to Asp₇₂₃contains 8 possible sites for N-linked glycosylation identified by thesequence Asn-X-Thr/Ser where X=any of the 20 amino acids with thepossible exception of aspartic acid. By analysis of the predictedsecondary structure of gB1, pellett et al. found 6 of 9 possibleglycosylation sites for gB1 on the surface of the protein at junctionsof helical or β-sheet structures and therefore likely to be efficientsubstrates for glycosylation. The remarkable amino acid homology betweenthe Type 1 and 2 proteins suggests that the utilization of potentialglycosylation sites is similar.

A comparison of the primary sequences of HSV-1 and HSV-2 glycoprotein Bis shown in FIG. 4. Amino acid differences between the Type 2 and Type 1proteins are highlighted by boxes. Overall the two proteins share anucleotide and an amino acid homology of 86%. However. the differencesappear to be significant, since only 12.5% of the amino acidsubstitutions between gB1 and gB2 are conservative changes. Thesedifferences in primary sequence are clustered in certain regions of theprotein resulting in long domains which are identical as well as smallregions of marked divergence.

The region of greatest divergence between gB1 and gB2 is the signalsequence. For gB2, the predicted signal sequence is only 22 amino acidsin length, as compared to 30 for gB1 strain Patton, and shares only 55%amino acid homology with the Type 1 protein. It is of interest to notethat while the length of the entire coding sequence for gB1 and gB2 isthe same (904 amino acids) the mature gB2 would be 7 amino acids longerthan gB1 due to its shorter signal peptide.

3.2 Expression of gB2 in mammalian cells

Expression of HSV-2 glycoprotein gB has been achieved in COS cells(transient expression) and in CHO cells (stable cell line secreting gB2)transformed with pHS210 alone or cotransformed with pHS210 and a secondplasmid containing dhfr.

Plasmid pHS210 was constructed as follows: The entire gene was subclonedas a 3.8 kb NruI-BamHI fragment in pBR322 to generate pHS208. See FIG.8. The PstI site at the 5' end of the gene, 100 bp to the right(downstream) of the NruI site, was changed to a HindIII site by in vitromutagenesis in M13. A HindIII to PvuII fragment of 1.9 kb was theninserted into pSV1, which was obtained by digestion of pSV1/dhfr withHindIII and BolII. See FIGS. 5 and 8. For this cloning step, pHS208 wascut with PvuII and the end repaired to blunt. The molecule was then cutwith HindIII and the 1.9 kb HindIII-(PvuII) fragment isolated by gelelectrophoresis. Likewise pSV1/dhfr was cut with Bg1II, repaired toblunt, cut with HindIII and the 4.85 kb HindIII-(BolII) vector fragmentisolated by gel electrophoresis. These two fragments (1.9 kb and 4.85kb) were ligated together to generate pHS210--the expression plasmid(FIG. 8).

Plasmid pHS210 was used directly to transform COS cells. Expression wasdetected by immunofluorescence using a gB specific monoclonal antibody,F3AB, and also using a commercially available polyclonal anti HSV-2antibody (DAKO) as the primary antibody screen. Secretion of gB2 intothe medium was detected by a gB2-specific ELISA. For this purpose,plates were coated with the monoclonal antibody. Samples of cell culturemedium were added to coated plates, then bound gB2 was detected with therabbit anti HSV-2 polyclonal antibody (DAKO) followed by horseradishconjugated goat antirabbit IgG.

For CHO cell transformation plasmid pHS210 was used along with a secondplasmid containing dhfr as a selective marker (FIG. 8) in acotransfection protocol. Following transfection and propagation inselective media, approximately 100 dhfr⁺ clones were isolated andscreened for synthesis and secretion of gB2 using an ELISA assay inwhich ELISA plates were coated with F3AB specific monoclonal antibody.Clone pHS210 #3-1, which had the highest levels of gB secretion, waschosen for further characterization of the gB2 polypeptide. The gB2protein was detected by labeling with [³⁵ S]-methionine followed byradio immunoprecipitation. After a 1 hr pulse, diffuse doublet bandscorresponding to polypeptides of 79 kd and 84 kd were detectedintracellularly. These proteins are larger than the 68,991 dalton sizepredicted for the 637 residue truncated gene product, and theypresumably correspond to partially glycosylated precursors. After a 5 hrchase, no gB2 was detected intracellularly, and an 89 kd polypeptide wasdetected in the medium. The size of the mature, fully glycosylated gB2secreted into the media of clone pHS210 #3-1 is somewhat smaller thanthe 100 kd gB1 secreted by pHS114-6 due to the removal from pHS210 ofthe coding sequence for 94 amino acids included in the gB1 plasmid.

4. Glycoprotein D1 4.1. Construction of yeast expression vectorscontaining synthetic DNA fragments coding for polypeptides A and B ofthe gD1 gene: pYHS109 and pYHS110 (FIG. 9).

Nucleotide seguences designated gD-A and gD-B based on portions of theamino acid sequence for glycoprotein D of HSV-1 reported by Watson etal. (1982) Science 218:381-384, and employing preferred yeast codons,were devised. The gD-A sequence, which encodes amino acids 253-283(corresponding to amino acids 258-288, as incorrectly numbered by Watsonet al., supra.) of the mature protein, was as follows: ##STR3##

The gD-8 sequence, which encodes amino acids 8-23 (corresponding toamino acids 13-28, as incorrectly numbered by Watson et al., supra.) ofthe mature protein, was as follows: ##STR4##

The sequences each include a KonI cohesive end at the 5'-end and a SalIcohesive end at the 3'-end. Coding for the mature secreted peptidebegins after the LysArg processing site. The 5'-end of each sequence isa modification of the 3'-end of the naturally occurring α-factorsecretory leader and processing signal sequence, where the modificationconsists of a deletion of three glu-ala pairs and a replacement of aleucine by a proline to create a KpnI site in the nucleotide sequence.The 3'-end of each sequence includes two translational stop codons, OPand OC.

Synthetic DNA fragments having the sequences just described wereprepared by synthesizing overlapping ssDNA segments as described byUrdea et al. (1984) Proc. Natl. Acad. Sci. USA 80:7461-7465 using thephosphoramidite method of Beaucage and Carruthers (1981) TetrahedronLett. 27:1859-1862, and annealing and ligating under the followingconditions.

The ssDNA fragments were joined as follows: 50 to 100 pmoles of eachsegment (except the two segments at the 5'-terminii) were5'-phosphorylated with 5.6 units of T4 polynucleotide kinase (NewEngland Nuclear) in 10 mM dithiothreitol (DTT), 1 mM ATP, 10 mM MgCl₂100 ng/ml spermidine, 50 mM Tris-HCl, pH 7.8 (total volume: 20 μl) for30 min at 37° C. Additional T4 kinase (5.6 units) was then added and thereaction continued for 30 min at 37° C. The fragments (except for the 5'terminii) were combined, diluted to 40 μl with water followed byaddition of 60 μl of 1M sodium acetate, 12 μl of 250 mM EDTA, and 5 μgof 1 mg/ml poly-dA. After heating for 5 min at 65° C., the 5' terminalpieces were added, followed by 420 μl of ethanol (100%). The solutionwas chilled for 20 min at -80° C., centrifuged, and the pellet waswashed twice with ethanol (100%) and dried. The pellet was redissolvedin water (18 μl ), heated to 100° C. for 2 minutes and then cooledslowly over 1.5 hours to 25° C. in a water bath.

The annealed fragment pool was ligated in a reaction mixture containingT4 DNA ligase (New England Biolabs, 1200 units) 1 mM ATP, 10 mM DTT, 10mM MgCl₂, 100 ng/ml spermidine, and 50 mM Tris-HCl, pH 7.8 (30 μl).After 1 hour at 14° C., the reaction mixture was partially purified bypreparative polyacrylamide gel (7%, native) electrophoresis. The DNA wasremoved from the appropriate gel slice by electroelution and ethanolcoprecipitation with poly-dA (5 μg).

After assembly, the synthetic gD-A and gD-B fragments were substitutedinto a KpnI/SaII-digested bacterial cloning plasmid pAB114αEGF-24, whichplasmid was prepared by cloning a mutagenized fragment of pYEGF-8 intopAB114 (see FIG. 1). The plasmids resulting from the insertion weredesignated pAB114αHS109 (gD-A) and pAB114αHS110 (gD-B).

The preparation of pAB114 was as follows: Plasmid pAB101 was obtainedfrom the screening of a random yeast genomic library cloned in YEp24(Fasiolo et al., 1981, J. Biol. Chem. 256:2324) using a synthetic 20-meroligonucleotide probe

    (5'-TTAGTACATTGGTTGGCCGG-3')

homologous to the published α-factor coding region (Kurjan andHerskowitz. Abstracts 1981, Cold Springs Harbor meeting on the MolecularBiology of Yeasts, page 242). Plasmid pAB1 l was obtained by deletingthe HindIII to SalI region of pBR322. An EcoRI fragment of into theunique EcoRI site in pAB1 l to produce pAB112. Plasmid pAB112 wasdigested to completion with HindIII, and then religated at low (4 μg/ml)DNA concentration to produce plasmid pAB113 in which three 63 bp HindIIIfragments were deleted from the α-factor structural gene, leaving only asingle copy of mature α-factor coding region. A BamHI site was added toplasmid pAB11 by cleavage with EcoRI, filling in of the overhanging endsby the Klenow fragment of DNA polymerase, ligation of BamHI linkers andreligation to obtain a plasmid designated pAB12. Plasmid pAB113 wasdigested with EcoRI, the overhanging ends filled in, and ligated toBamHI linkers. After digestion with BamHI, the resulting 1500 bp whichcarries the single copy of the α-factor gene fragment was gel-purifiedand ligated to pAB12 which had been digested with BamHI and treated withalkaline phosphatase to produce pAB114, which contains a 1500 bp BamHIfragment carrying the α-factor gene.

The preparation of pYEGF-8 was as follows: A synthetic sequence forhuman epidermal growth factor (EGF) was prepared and ligated to pAB112(described above) which had been previously completely digested withHindIII and SalI to produce pAB201. The HindIII site lies within the3'-end of the α-factor gene, and the EGF sequence was inserted usingappropriate linkers. The resulting plasmid was designated pAB201.

Plasmid pAB201 (5 μg) was digested to completion with EcoRI and theresulting fragments were filled in with DNA polymerase I Klenow fragmentand ligated to an excess of BamHI linkers. The resulting 1.75 kbpfragment was isolated by preparative gel electrophoresis, andapproximately 100 ng of this fragment was ligated to 10 ng of yeastplasmid pCl/l (described below) which had been previously digested tocompletion with restriction enzyme BamHI and treated with alkalinephosphatase. The ligation mixture of the 1.75 kbp fragment carrying thepartial α-factor gene fused to the EGF gene and pCl/l was used totransform E. coli HB101 cells, and transformants were selected based onampicillin resistance. DNA from one ampicillin resistant clone(designated pYEGF-8) was used to transform yeast AB103 (genotype: MATα.pep 4-3, leu 2-3, leu 2-112, ura 3-52, his 4-580, cir° ) cells, andtransformants selected based on their leu⁺ phenotype.

Plasmid pCl/l is a derivative of pJDB219 (Beggs (1978) Nature 275:104)where the region derived from bacterial plasmid pMB9 has been replacedby pBR322. The pCl/l plasmid carries genes for both ampicillinresistance and leucine prototrophy.

Plasmid pAB114αEGF-24 was generated by an in vitro mutagenesis procedurewhich deleted the sequences coding for the glu-ala processing region inthe α-factor leader. Plasmid pAB114αEGF-24 was obtained as follows: aPstI-SalI fragment of pYEGF-8 containing the α-factor leader hEGF fusionwas cloned in phage M13 and isolated in single-stranded form. Asynthetic 36-mer oligonucleotide primer

    (5'-GGGGTACCTTTGGATAAAAGAAACTCCGACTCCGAA-3')

was used as a primer for the synthesis of the second strand using theKlenow fragment of DNA polymerase I. After fill-in and ligation at 14°C. for 18 hours, the mixture was treated with S₁ nuclease and used totransfect E. coli JM101 cells. Phage containing DNA sequences in whichthe glu-ala region was removed were located using ³² p-labeled primer asa probe. DNA from positive plaques was isolated, digested with PstI andSalI, and the resulting fragment inserted into pAB114 (described above)which has been previously digested to completion with SalI, partiallywith PstI and treated with alkaline phosphatase. The resulting plasmidwas designated pAB114αEGF-24.

Referring again to FIG. 9, the BamHI-BamHI fragment of pAB114αHS109 orpAB114αHS110 (1588 base pairs for gD-A and 1546 base pairs for gD-B) wasexcised and ligated into the unique BamHI site of pCl/l. The resultingexpression vectors were designated pYHS109 for gD-A and pYHS110 forgD-B.

4.2 Expression of gD-A and gD-B polypeptides in yeast

Plasmids pYHS109 and pYHS110 were both used to transform yeast strainAB103.1 (a, pep 4-3, leu 2-3, leu 2-112, ura 3-52, his 4-580, cir°) toleu prototrophy following the procedure of Hinnen et al., Proc. Natl.Acad. Sci. USA (1978) 75:1929-1933. The transformants were grown in 1 Lcultures at 30° C. in buffered leucine-deficient media to saturation,corresponding to an absorbance of 5 at 650 nm. Yeast cell cultures weremaintained at saturation for an additional 12 to 24 hrs with shaking at30° C. The cultures were then harvested, the intact yeast cells pelletedby centrifugation at 3000 RPM, and the resulting supernatant mediafiltered through a 0.22 μ Millipore filter. This fraction was thenpassed through a C18 reverse phase column, constructed from 8 Seppakunits purchased from Waters. The bound material was eluted with 30 ml of80% (v/v) acetonitrile, 0.1% (v/v) trifluoroacetic acid in water,evaporated to dryness with a Buchii RotoVap and redissolved in 1.6 ml ofdistilled water. This material was separated on an HPLC C18 columnmonitored at 210 nm. The peak corresponding to each respective peptidewas collected and its identity confirmed by antigenicity. Each peptidereacted specifically in an ELISA assay using rabbit polyclonal antiserawhich has been raised against a chemically synthesized gD-B peptide orpartial gD-A peptide (residues 256 through 271 of the sequence shown forgD-A in page 9) purchased from Vega Biochemicals. Expression levels, asdetermined by spectrophotometric measurements of HPLC purified peptides,were on the order of 7.6 mg of gD-A per liter of yeast culture (OD₆₅₀=5) and 0.6 mg of gD-B per liter of yeast culture (OD₆₅₀ =5). Theseresults demonstrate the feasibility of expressing a relatively shortportion or fragment of a protein and its secretion from yeast cellsusing an α-factor expression vector.

4.3 Construction of yeast vectors for high level intracellularexpression using fragments of the naturally occurring gD1 gene

Nucleotide fragments from the naturally occurring gD gene expressing gDof HSV-1 (gD1) were also expressed intracellularly in yeast undercontrol of the GAPDH promoter and terminator. A library of EcoRIfragments of HSV-1, strain Patton, cloned into the EcoRI site of pBR322,was made by Dr. Richard Hyman, Hershey Medical Center, Hershey, Pa. ThegD is entirely contained within a 2.9 kb SacI fragment within the EcoRIfragment of one clone (clone H) isolated from the HSV-1 library. Clone Hwas obtained from Dr. Hyman; the 2.9 kb fragment was purified by gelelectrophoresis and was used for the construction of several expressionvectors which differ in the size of the gD fragment cloned and/or thesynthetic linkers used in the 5' or 3' ends of the gD fragments. FIG. 10illustrates the protein coding region (boxed region) and the fragmentsused for the construction of yeast expression vectors pYHS115, pYHS116,pYHS117, pYHS118 and pYHS119. A description of the construction of eachplasmid follows.

4.3.1 Construction of pYHS115

Plasmid pYHS115 contains the gD gene in a GAPDH expression cassettecloned into the BamHI site of pCl/l (described hereinabove).

The GAPDH expression cassette was constructed as follows: Threefragments were prepared as described in detail below):

(a) A BamHI-HindIII fragment (1407 bp) containing 346 bp of pBR322 and1061 bp of the GAPDH promoter;

(b) A HindIII-SalI fragment (1430 bp) containing the gD gene, and

(c) A SalI-BamHI fragment (900 bp) containing the GAPDH terminator.

These fragments were ligated together and the mixture was digested withBamHI. The 3.7 kb resulting cassette was isolated by gel electrophoresisand ligated to BamHI cut, alkaline phosphatase-treated pCl/l (FIG. 11).

Fragment (a) was prepared by completely digesting pGAP347 (describedbelow) With BamHI followed by partial digestion with HindIII. Theresulting 1407 bp fragment containing 346 bp of pBR322 and 1061 bp ofthe GAPDH promoter was isolated by gel electrophoresis.

Construction of pGAP347 was as follows. PolyA⁺ RNA was isolated from S.cerevisiae yeast strain A364A. Double-stranded cDNA was synthesizedusing AMV reverse transcriptase and E coli DNA polymerase I.Poly-dC-tails were added to the double-stranded cDNA molecule usingdeoxynucleotide terminal transferase. Poly-dC-tailed cDNA was annealedto poly-dG-tailed pBR322 and used to transform E. coli HB101. 1000transformants were screened by colony hybridization to labeled PolyA⁺RNA, and a subset further examined by restriction endonuclease mapping,and DNA sequencing. Three clones containing GAPDH sequences wereisolated from the the pool. One clone (pcGAP-9) contained an insert ofabout 1200 base pairs and was used for further work.

A yeast gene library was prepared by inserting fragments obtained afterpartial digestions of total yeast DNA with restriction endonucleaseSau3A in lambda-phage Charon 28, according to Blattner et al., Science(1977) 196:161-169. Several fragments containing yeast GAPDH codingsequences were isolated by screening the phage library with labeled DNAfrom pcGAP-9. The yeast GAPDH gene of one of these clones was subclonedin pBR322 as a 2.1 kb HindIII fragment (pGAP-1). The GAPDH promoterregion was isolated from these clones. A HhaI-HindIII fragment of about350 bp containing the 3' portion of the promoter was obtained by: a)digestion of pGAP-1 with HinfI to generate an approximately 500 bpsegment Which includes the 3' part of the promoter and a region encodingthe N-terminal amino acids of GAPDH; b) resection with Ba131 to yield a400 bp fragment lacking the GAPDH coding region (3'-terminus one baseupstream from the ATG initiator codon); c) addition of HindIII linkers;and d) cleavage with HhaI. A second HindIII-HhaI fragment of about 700bp containing the 5' portion of the promoter was isolated from pGAP-1,ligated to the 350 bp HhaI-HindIII fragment and treated with HindIII.The resulting 1061 bp HindIII fragment was isolated by gelelectrophoresis and cloned in HindIII digested, alkalinephosphatase-treated pBR322 to produce pGAP347.

Fragment (b) was obtained as follows: Clone H, isolated from the HSV-1Patton library, was digested with SacI. A 2.9 kb SacI fragment waspurified by gel electrophoresis and subsequently digested with HindIIIand NruI. The 1430 bp HindIII-NruI fragment containing the gD gene (FIG.3) was purified by gel electrophoresis, ligated to NruI-SalI adaptors ofthe following sequence: ##STR5## and digested with SalI.

Fragment (c) was obtained as follows: A 900 bp fragment containing theGAPDH terminator was obtained by BamHI and SalI digestion of pUH28(described under "Construction of pYHS117") and purification by gelelectrophoresis.

4.3.2 Construction of pYHS116

pYHS116 contains a gD gene fragment which has a 600 bp deletion at the5' end of the coding region that comprises most of the signal sequencecoding region. To construct pYHS116, two fragments were obtained:

(a) A BamHI-HindIII fragment (1407bp) containing 346 bp of pBR322 and1061 bp of the GAPDH promoter. This fragment was obtained as describedunder "Construction of pYHS115."

(b) A NcoI-BamHI fragment (2150 bp) containing the partial gD genefollowed by the GAPDH terminator. This fragment was obtained byBamHI/NcoI digestion of pYHS115 (described previously) and purificationby gel electrophoresis. A HindIII-NcoI chemically synthesized adaptor ofthe following sequence: ##STR6## was ligated to the fragment. Thisadaptor provides for the first two codons (met and val) fused in thecorrect reading frame to the partial gD.

Fragments (a) and (b) were ligated together and subsequently digestedwith BamHI. The resulting 3.5 kb cassette was isolated by gelelectrophoresis and ligated to BamHI cut, alkaline phosphatase-treatedpCl/l.

4.3.3 Construction of pYHS117.

Plasmid pYHS117 contains the same partial gD gene clone in pYHS116,fused in reading frame to 7 extra codons at the 5'-end, which code forthe first 7 amino acids of the GAPDH structural gene. To constructpYHS117 two fragments were obtained.

(a) An NcoI-SalI digested vector (6.8 kb) comprising pBR322 sequences,the GAPDH promoter fused to the first 7 codons of the structural geneand the GAPDH terminator. This vector was prepared by NcoI digestion ofpUH28 (described below), followed by a partial digestion with SalI andpurification by gel electrophoresis.

(b) An Ncol-SalI fragment (1430 bp) containing a partial gD gene. Thisfragment was obtained by NcoI-SalI digestion of pYHS115 (describedpreviously) and purification by gel electrophoresis.

These two fragments were ligated together to yield a pBR322 derivedvector which contains a partial gD gene fused in reading frame to the 7first codons of GAPDH gene, flanked by the GAPDH promoter in its 5' endand by the GAPDH terminator in its 3' end. The gD expression cassettewas obtained by digesting this plasmid with BamHI and purifying a 3.4 kbfragment by gel electrophoresis. This fragment was ligated to BamHIdigested, alkaline phosphatase-treated pCl/l to produce pYHS117.

Plasmid pUH28 contains the coding and 3' noncoding regions of thehepatitis B surface antigen (HBsAg) gene fused in incorrect readingframe to the first 7 codons of the GAPDH structural gene. This fusion isflanked in its 5' end by the GAPDH promoter and in its 3' end by part ofthe GAPDH coding region followed by the GAPDH terminator. This plasmidwas constructed so as to have an NcoI site at the 3' end of the first 7codons of the GAPDH gene with the following sequence: ##STR7## When thisNcoI end is ligated to the partial gD fragment (b, described above) thecorrect reading frame for the gD protein is regenerated. The SalI siteused in the preparation of fragment a (described above) is at the 5'region of the GAPDH terminator. Therefore, a deletion of the sAg codingplus noncoding regions and GAPDH coding region was obtained by digestingpUH28 with NcoI and partially with SalI.

The construction of pUH28 involves cloning of fragment that contains theHBsAg coding and 607 bp of 3' noncoding regions prepared from pHBS5-3Hae2-1 (described below) into the GAPDH containing vector pGAP₂(described below). To prepare the fragment, pHBS5-3 Hae2-1 waslinearized by PstI digestion, partially digested with NcoI and aPst-NcoI fragment of 1.9 kb containing pBR322 sequences. HBsAg codingand 3' sequences was purified by gel electrophoresis. This fragment wassubsequently digested with EcoRI and a 1.2 kb NcoI-EcoRI fragmentcontaining the HBsAg coding and 3' noncoding regions was purified by gelelectrophoresis. Plasmid pGAP₂ was linearized with XbaI and treated withBal31 to remove approximately 100 bp. The plasmid was subsequentlydigested with NcoI and a vector fragment of about 9 kb was purified bygel electrophoresis. The NcoI ends of the vector and the 1.2 kbNcoI-EcoRI fragment encoding HBsAg were ligated. The recessed end wasfilled in with Klenow and the resulting blunt end was ligated to theblunt produce pUH28.

pHBS5-3 Hae2-1 is a plasmid that contains the HBsAg coding regions and607 bp of 3' flanking sequences. This plasmid is a derivative of pHBS5-3which contains the same insert but only 128 bp of 3' untranslated regioninstead of 607 bp. Plasmid pHBS5-3 has been previously described incopending U.S. application Ser. No. 609,540, filed May 11, 1984 (pp.13-14), which disclosure is incorporated herein by reference. pHBS5-3Hae2-1 was constructed as follows. The HBV genome (3.2 kb) was excisedfrom pHB-3200 (Valenzuela et al., Nature (1979) 280:815-819) byrestriction digestion with EcoRI. The 3.2 kb fragment was purified bygel electrophoresis and was recircularized by ligation of the EcoRIsticky ends. This closed HBV genome was digested with HaeII, which cutsin the 3' noncoding regions. Recessed ends were filled in with Klenowand HindIII linkers were ligated. The DNA was cut with HindIII andsubsequently with XbaI, which has a single site in the HBS codingregion. A 1.2 kb XbaI-HindIII fragment containing 586 base pairs of thecoding sequence of HBV and 607 base pairs of the 3' noncoding region wasisolated by gel electrophoresis. This fragment was cloned into pHBS5-3previously cut with XbaI and HindIII and treated with alkalinephosphatase, to yield pHBS5-3 Hae2-1.

pGAP-2 is a pBR322 derived vector which contains a BamHI insert that hasthe GAPDH coding sequence. 5' and 3' flanking regions. There are twoXbaI sites in this plasmid: one in the coding region and one in the 3'flanking sequences. pGAP-2' is a derivative of pGAP-2 in which the XbaIsite present in the 3' flanking regions has been eliminated. For thispurpose, 50 μg of pGAP-2 were partially digested with XbaI, treated withBal31 to remove 25 base pairs per end, and ligated. The plasmids wereused to transform E. coli HB101 and the transformants were selected forloss of XbaI site in the 3' flanking region.

4.3.4. Construction of pYHS118

This vector contains a partial gD gene with deletions in two regions: a600 bp deletion in the 5'-end coding regions which comprises most of thesignal sequence coding region and a 1300 bp deletion in the 3'-endcoding region which includes most of the anchor sequence coding region.It also contains 7 extra codons from the GAPDH gene coding regions fusedin the reading frame at the 5' end of the gD gene, similar to pYHS117.Plasmid pYHS118 was constructed as follows: pYHS115 was digested withNcoI and SalI, the resulting 1430 bp fragment containing the partial gDwas purified by gel electrophoresis and submitted to digestion with NarI(FIG. 10). The two resulting fragments (fragment a: 873 bp containing 5'end and fragment b: 411 bp containing 3' end) were independentlyisolated by gel electrophoresis. Fragment b was subsequently digestedwith Sau96A to yield three fragments which were separated by gelelectrophoresis. The 87 bp Nar-Sau96A fragment was recovered from thegel and was ligated to Sau96A-SalI synthetic adaptors of the followingsequence: ##STR8## The NarI-(Sau96A)SalI fragment (102 bp) was digestedwith SalI, purified by gel electrophoresis and ligated with fragment a(previously described). The resulting NcoI-SalI fragment (975 bp) wasligated to NcoI-SalI, digested and gel purified pUH28 as described under"Construction of pYHS117." The resulting pBR322 derived vector wasdigested with BamHI and a 3.1 kb fragment containing the gD expressioncassette was purified by gel electrophoresis. This cassette was ligatedto BamHI digested, alkaline phosphatase treated PCl/l to producepYHS118.

4.3.5 Construction of pYHS119.

This vector contains a partial gD gene with deletions in two regions: a600 bp deletion in the 5' end coding regions which comprises most of thesignal sequence coding regions and a 2400 bp deletion in the 3' endcoding regions which includes all the anchor sequence coding regions andabout 700 bp upstream of the anchor sequence. It also contains 7 extracodons from the GAPDH gene coding region fused in reading frame at the5' end of the gD gene, as pYHS117 and pYHS118. Plasmid pYHS119 wasconstructed as follows: pYHS115 was digested with NcoI and SalI, theresulting 1430 bp fragment containing the partial gD was purified by gelelectrophoresis and subsequently digested with NarI. The 873 bpNcoI-NarI fragment was isolated by gel electrophoresis. A syntheticadaptor of the following sequence: ##STR9## which provides complementarynucleotides to the NarI 5' overhang, 3 codons in reading frame, a stopcodon and a 5' overhang of SalI, was ligated to the 873 bp NcoI-NarIfragment then digested with SalI. The resulting NcoI-SalI fragment wasligated to pUH28 which had been previously completely digested with NcoIand partially digested with SalI and purified by gel electrophoresis asdescribed under "Construction of pYHS117." The resulting pBR322 derivedvector was digested with BamHI and a 2.2 kb fragment containing the gDexpression cassette was purified by gel electrophoresis. This cassettewas ligated to BamHI digested, alkaline phosphatase-treated pCl/l toproduce pYHS119.

4.4 Synthesis of gD1 from yeast vectors containing partial or completegD1 gene

Plasmids pYHS115, 116, 117, 118 and 119 were used to transform yeaststrain AB103.1 (a, pep 4-3, leu 2-3, leu 2-113, ura 3-52, his 4-580,cir°) following the procedure of Hinnen et al., supra. Thetransformations were grown to an OD₆₅₀ =3 at 30° C. in YEPD media. Thecultures were then harvested by pelleting the yeast cells at 3000 RPM.Cells were spheroplasted with zymolyase and subsequently osmoticallylysed in a hypotonic solution. Membranes were spun down in an Eppendorfcentrifuge, and the pellet was solubilized in 0.1% SDS with proteaseinhibitors for 16 hours at 4° C. The suspension was centrifuqed andtotal protein, as well as gD specific protein, was determined in bothsoluble and insoluble fractions. Expression of the gD gene in each ofthe above described constructions was detected by Western Blothybridization (Towbin et al., Proc. Natl. Acad. Sci. USA (1979)76:4350). For this purpose protein samples were submitted toSDS-polyacrylamide gel electrophoresis (Laemmli, Nature (1970 227:680)and electroblotted onto nitrocellulose filters (Towbin et al., supra.).The filter was preincubated with goat serum and subsequently treatedwith a rabbit polyclonal antibody raised against HSV-1 (Dako). Thefilter was then incubated with a second goat anti-rabbit antibodyconjugated with horseradish peroxidase (Boehringer-Mannheim) and finallyit was incubated with horseradish peroxide color development reagent(Bio-Rad) and washed. The results indicate that immunoreactive materialis being synthesized in yeast AB103.1 strain transformed with gDexpression vectors, with the exception of transformants containingpYHS115. In all other cases, gD protein corresponds to 0.1 to 0.5% oftotal yeast cell protein.

4.5 Construction of mammalian expression vectors for gD1: pHS132 (FIG.12).

A library of EcoRI fragments of HSV-1, strain Patton, cloned into theEcoRI site of pBR322 was made by Dr. Richard Hyman, Hershey MedicalCenter, Hershey, Pa. The gD1 gene is entirely contained within a 2.9 kbSacI fragment within the EcoRI fragment of clone H from this library.Clone H, containing a 15 kb EcoRI insert, was obtained from Dr. Hyman.The 2.9 kb fragment was purified by gel electrophoresis and thendigested to completion with HindIII and NcoI. The 5' end of the gD gene,consisting of 74 bp of 5' untranslated sequences plus 60 bp coding forthe amino terminal 20 amino acids. was gel isolated as a 134 bpfragment. The 3' end of the gD gene was obtained by digestion of pHYS119(see Section 4.3.5) with NcoI and SalI and isolation of the 873 bpfragment. These two fragments (5' and 3' ends) were ligated togetherwith the plasmid pUC12 which had previously been digested with HindIIIand SalI. The pUC12 vector is commercially available from Pharmacia andP-L Biochemicals, the resulting plasmid was designated pHS131. Theplasmid pHS131 was digested with HindIII, the 5'-4 base pair overhangwas filled in with Klenow polymerase and then digested with SalI. The1007 bp fragment containing the gD gene was gel isolated and ligatedinto the plasmid pSV7d which had previously been cut with SmaI plusSalI. The plasmid pSV7d is described below. The resulting expressionvector is designated pHS132. Its derivation is outlined in FIG. 12.

The plasmid encodes 315 amino acids of gD1 protein including a 25 aminoacid signal sequence out of a total of 399 amino acids for the completeprotein. The protein has been truncated at the carboxyl terminus andlacks 84 amino acids including the hydrophobic membrane anchor domainand the cytoplasmic domain such that the resulting protein is secretedinto the medium.

The plasmid pSV7d was constructed as follows: the 400 bp BamHI/HindIIIfragment containing the SV40 origin of replication and early promoterwas excised from SVgtI (Mulligan, R., et al., J. Mol. Cell Biol. (1981)1:854-864) and purified. The 240 bp SV40 BclI/BamHI fragment containingthe SV40 poly A addition site was excised from pSV2/dhfr (Subramani etal., J. Mol. Cell Biol. (1981) 1:854-864) and purified. The fragmentswere fused through the following linker: ##STR10## This linker containsfive restriction sites, as well as stop codons in all three readingframes. The resulting 670 bp fragment (containing the SV40 origin ofreplication, the SV40 early promoter, the polylinker with stop codonsand the SV40 polyadenylation site) was cloned into the BamHI site ofPML, a pBR322 derivative with about a 1.5 kb deletion (Lusky andBotchan, Cell (1984) 36:391). to yield PSV6. The EcoRI and EcoRV sitesin the PML sequences of PSV6 were eliminated by digestion with EcoRI andEcoRV, treated with Bal31 nuclease to remove about 200 bp on each end,and finally religated to yield PSV7a. The Bal31 resection alsoeliminated one BamHI restriction site flanking the SV40 region,approximately 200 bp away from the EcoRV site. To eliminate the secondBamHI site flanking the SV40 region, pSV7a was digested with NruI, whichcuts in the pML sequence upstream from the origin of replication. Thiswas recircularized by blunt end ligation to yield pSV7 b.

pSV7c and pSV7d represent successive polylinker replacements. Firstly,pSV7 b was digested with StuI and XbaI. Then, the following linker wasligated into the vector to yield pSV7c: ##STR11## Thereafter, pSV7c wasdigested with BglII and XbaI, and then ligated with the following linkerto yield pSV7d: ##STR12##

4.6 Expression of gD1 in mammalian cells

Expression of gD1 from plasmid pHS132 has been demonstrated in manyexperiments. First, specific immunofluorescence was observed in COS 7cells following transfection using the methods described previously andusing a commercially available rabbit sera against HSV-1 (DAKO) fordetection. Second, stable CHO cell lines secreting gD1 were established.The expression levels were analyzed by ELISA and verified byradioimmunoprecipitation of pulse labeled and chased cell lysates andmedia. Third, gD1 was purified from the media of roller bottle culturesof the CHO cell line D64 by sequential steps of ammonium sulfateprecipitation. immunoaffinity chromatography and ultrafiltration. Forthe affinity chromatography the gD monoclonal antibody 8D2 described inRector et al. (1982) supra, linked to cyanogen bromide activatedSepharose 4B was employed.

5. Glycoprotein D2 5.1 Construction of mammalian expression vectors forgD2

The HindIII L fragment of HSV-2 strain 333 was cloned in pBR322 by Dr.Richard Hyman as noted in the reference Kudler et al., Virology (1983)124:86-99. The gene for the glycoprotein gD2 had been mapped to theshort unique region of the virus between 0.90-0.945 map units byRuyechan et al., J. Virol. (1970) 29:677-697, a region covered by theHindIII L fragment as shown in the genomic map of Roizman, B., Ann. Rev.Genet (1979) . 13:25-57. The DNA sequence of the gD2 gene has beenpublished by Watson, Gene (1983) 26:307-312.

The HindIII L fragment cloned in pBR322 was obtained from Dr. RichardHyman and the restriction map shown in FIG. 13A determined. The gene forgD2 was found to lie on a 2.4 kb XhoI fragment by probing Southern blotsof restriction digests of the HindIII L fragment with the 2.9 kb SacIfragment encoding gD1. A map of the XhoI fragment and the position ofthe gD2 gene is shown in FIG. 13B. The 2.4 kb XhoI fragment was clonedin a pBR322 derivative vector containing an XhoI site to generateplasmid pHS204. Three different gD2 expression vectors, plasmids pHS211,pHS212, and pHS213 were constructed as follows and as diagrammed in FIG.14. The plasmid pHS211 encodes the first 305 amino acids of gD2including the signal sequence. For its construction pHS204 was cut withSmaI and BamHI and two restriction fragments were gel isolated: a 250 bpSmaI fragment containing the 5' end of the gene including 82 bp of 5'untranslated sequence and the 3' adjacent 746 bp SmaI-BamHI fragmentcontaining an interior portion of the gene. The mammalian cellexpression vector pSV7d (described in Section 4.5) was cut with EcoRI,the 5' 4 bp overhang repaired to blunt with Klenow polymerase and thencut with BamHI. The two fragments from pHS204 were ligated into thedigested pSV7d and bacterial transformants were screened for theappropriate orientation of the SmaI fragment to generate the vectorpHS211.

The plasmid pHS212 which encodes 352 amino acids of gD2 or 47 additionalresidues beyond those present in pHS211 was constructed by the digestionof pHS204 with HaeII and repairing the ends to blunt with Klenowpolymerase followed by digesting with BamHI. A 141 bp (HaeII) (theparenthesis intends the terminus has been filled in) to BamHI fragmentwas gel isolated. The plasmid pHS211 was transferred into the E. colistrain GM272 (dam⁻) and plasmid DNA prepared, which was then restrictedwith BclI followed by blunt end repair with Klenow polymerase thendigestion with BamHI. The large vector fragment (about 3.4 Kb) was gelisolated and ligated together with the 141 bp (HaeII)-BamHI fragment togenerate the plasmid pHS212. The fusion of gD2 sequences to plasmidvector sequences at the 3' end of the gene results in the addition of 27codons of nonsense DNA to the 3' end of the gD2 gene. To eliminate thesenonsense sequences the plasmid pHS213 was constructed by partialdigestion of pHS211 with SalI and gel isolation of the single cutplasmid which was then repaired to blunt with Klenow polymerase anddigested with BamHI. The (HaeII) to BamHI fragment of 141 bp from pHS204was ligated into the linearized, pHS211 to generate the plasmid pHS213.

5.2 Expression of gD2 in mammalian cells

The expression of gD2 in mammalian cells was first assayed bytransfection of COS 7 cells with pHS211, pHS212 and pHS213 for transientexpression. Expression of gD2 was detected both by immunofluorescenceand by capture ELISA analysis of COS 7 conditioned media using a rabbitanti-HSV-2 antibody for the immunofluorescence and a gD type commonantibody, 8D2 (Rector et al., (1982) supra.), for the capture antibodyin the ELISA.

Permanent CHO cell lines were then established by transfection with theplasmids pHS211 or pHS213 with Ad dhfr and selection for dhfracquisition and screening by ELISA for gD2 expression.

Description of Ad-dhfr

The plasmid bearing the dhfr gene was constructed by fusing the majorlate promoter from adenovirus-2 (Ad-MLP, map units 16-17.3) to the mousedhfr cDNA at the 5' end. DNA coding for the intron for SV40 small tAntigen and the SV40 early region polyadenylation site was obtained frompSV2-neo, described in Southern and Berg, J. Mol. Appl. Genet. (1982)1:327-341, and fused to the 3' end of the dhfr cDNA. These threesegments were subcloned into pBR322 to obtain the plasmid Ad-dhfr. Thisplasmid is functionally similar to the dhfr plasmid described in Kaufmanand Sharp, Molec. and Cell Biol., (1982) 2:1304-1319.

6. Vaccine Studies 6.1 Protection of guinea pigs against initial andrecurrent genital herpes: immunization prior to infection with HSV-2

The recombinant gB1 protein was produced as described in Section 2.2above. The gB protein was purified by sequential steps of lentil lectinchromatography, immunoaffinity chromatography, and concentration byultrafiltration resulting in a preparation which was 70% homogeneous asdetermined by SDS polyacrylamide gel electrophoresis.

The recombinant gD1 protein was prepared as described in Sections 4.5and 4.6.

Total HSV-2 glycoproteins were prepared HSV-2 strain 333 infected Verocells by lentil lectin Sepharose chromatoqraphy using the method ofRespess et al. J. Virol. Methods (1984) 8:27. These mixtures containedapproximately 15% gB and 4.5% gD, as well as high concentrations of gC,lower amounts of gE and gG plus a mixture of unidentified HSV and Verocell proteins.

This study was designed to test the effect of a variation in route ofimmunization, adjuvant (complete Freund's adjuvant versus alum) and theefficacy of recombinant mammalian gD plus recombinant mammalian gB.Eighty-one female Hartley guinea pigs were immunized as noted in thefollowing table.

    ______________________________________                                        Group Treatment     Dose     Adjuvant                                                                             Route N                                   ______________________________________                                        2     CHO cell extract                                                                             8 μg Alum   SQ*   12                                  3     HSV-2 total   50 μg Freund's                                                                             Foot- 9                                         glycoprotein (pG2)            pad                                       4     pG2           50 μg Alum   Foot- 9                                                                       pad                                       5     pG2           50 μg Alum   SQ    8                                   6     HSV-1 gB      40 μg Alum   SQ    8                                   7     HSV-1 gD      40 μg Alum   SQ    9                                   8     HSV-1 gB + gD 40 μg +                                                                             Alum   SQ    8                                                       40 μg                                                  1     Untreated      --       --     --   18                                  ______________________________________                                         *Subcutaneously                                                          

Group 2 was immunized twice on days -63 and -28. All other groups wereimmunized thrice on days -58, -42 and -21. On day 1 all pigs wereintravaginally inoculated with 5×10⁵ Pfu HSV-2 strain MS. Groups 6-8 arerecombinant mammalian produced glycoproteins. The course of the initialgenital HSV-2 infection was evaluated as before with the results shownin Table 1 below. The experiment shows that the choice of both route andadjuvant modifies the outcome of the primary disease; alum is a lesseffective adjuvant than complete Freund's adjuvant for these antigensand the subcutaneous route is less effective than the footpad. Moreimportantly for the present application, the mixture of recombinant gB1plus gD1 affords better protection than the mixture of glycoproteinsfrom HSV-2 infected Vero cells (compare groups 5 and 8). The pattern ofrecurrent disease for these same animals is shown in Table 2 and theconclusion are essentially the same as noted for primary disease.

                                      TABLE 1                                     __________________________________________________________________________    Effect of HSV glycoprotein vaccines on primary HSV-2 genital infection in     guinea pigs.                                                                                            Animals                                                                            Severity                                                                            Duration                                            Dose           with Skin                                                                          of Skin                                                                             of Urinary                                                                          %                                  Group                                                                             Treatment.sup.a                                                                      μg                                                                              Adjuvant.sup.b                                                                      Route.sup.c                                                                       Lesions                                                                            Disease.sup.d                                                                       Retention.sup.e                                                                     Mortality.sup.f                    __________________________________________________________________________    1   None   --   --    --  19/19                                                                              14.8 ± 1.0                                                                       5.4 ± 0.4                                                                        32                                 2   CHO Extract                                                                           8   Alum  SQ  11/11                                                                              11.9 ± 1.4                                                                       5.3 ± 0.3                                                                        27                                 3   gP2    50   CFA   FP  0/6  0     0     0                                  4   gP2    50   Alum  FP  4/9  0.7 ± 0.5                                                                        0.6 ± 0.6                                                                        0                                  5   gP2    50   Alum  SQ  5/8  2.3 ± 0.9                                                                        3.0 ± 1.0                                                                        22                                 6   gB     40   Alum  SQ  7/8  2.8 ± 1.1                                                                        3.9 ± 0.7                                                                        0                                  7   gD     40   Alum  SQ  7/8  2.1 ± 1.0                                                                        2.5 ± 0.8                                                                        0                                  8   gB + gD                                                                              40 + 40                                                                            Alum  SQ  8/8  1.7 ± 0.7                                                                        1.5 ± 0.6                                                                        0                                  __________________________________________________________________________     .sup.a Vaccines administered 9.6 and 3 was prior to intravaginal HSV2         inoculation with 5.3 log.sub.10 pfs HSV2 (MS Strain) except group 3 which     was immunized 9 and 4 weeks prior to viral challenge.                         .sup.b Alum = Aluminum phosphate (10%); CFA = complete freund's adjuvant.     .sup.c SQ = Subcutaneously in hindlimb; FP hindlimb footpad.                  .sup.d Mean area under the skin lesion scoreday curve ± error.             .sup.e Mean days ± standard error.                                         .sup.f Deaths within 14 days of HSV2 inoculation.                        

                  TABLE 2                                                         ______________________________________                                        Effect of HSV glycoprotein vaccination on the pattern of                      recurrent genital HSV-2 infection in guinea pigs.sup.a                                               Days Lesions                                                                           Recurrent                                                            Observed.sup.b                                                                         Episodes.sup.c                                                       (Mean ±                                                                             (Mean ±                                                                            Days/                                 Group Treatment  N     SE)      SE)     Episodes                              ______________________________________                                        1 & 2 Control    9     20.6 ± 2.4                                                                          10.9 ± 1.2                                                                         1.9                                   3     gP2/CFA/   6      3.2 ± 1.3                                                                          2.3 ± 1.0                                                                          1.4                                         Footpad                                                                 4     gP2/Alum/  8      3.0 ± 0.8                                                                          2.4 ± 0.6                                                                          1.3                                         Footpad                                                                 5     gP2/Alum/  5     11.8 ± 2.0                                                                          7.0 ± 0.9                                                                          1.7                                         SQ                                                                      6     gB/Alum/SQ 6     13.8 ± 2.4                                                                          7.5 ± 1.4                                                                          1.8                                   7     gD/Alum/SQ 9     11.2 ± 2.1                                                                          7.4 ± 1.2                                                                          1.5                                   8     gB + gD/   8      9.9 ± 1.8                                                                          5.9 ± 0.9                                                                          1.7                                         Alum/SQ                                                                 ______________________________________                                         .sup.a Animals examined for recurrent lesions day 14-92 after intravagina     HSV2 challenge.                                                               .sup.b All groups except gP2/Alum/SQ significantly different from control     (p < 0.05).                                                                   .sup.c All groups except gD significantly different from control (p <         0.05).                                                                   

6.2. Therapeutic Studies: Effect of Recombinant HSV glycoproteinvaccines administered after primary infection on subsequent recurrentherpetic diseases in guinea pigs

Female Hartley guinea pigs were intravaginally inoculated with 5×10⁵ pfuHSV-2 MS strain on day 1. Animals were treated with acyclovir (5 mg/ml)from days 1-10 by addition to the drinking water. Acyclovir reduces theseverity of primary infection and thus the mortality, incidence ofsecondary bacterial infection and incidence of genital scarring. The useof acyclovir during primary infection has been shown to have no impacton the course of the disease after the cessation of treatment for theguinea pig (Bernstein et al., Virology, (1986) 67:1601). After recoveryfrom primary infection, the animals were immunized with HSV-2 totalglycoprotein preparation (gP2). with a mixture of recombinant gB1 andgD1 (HSV-1 gB+gD) or received no treatment. Treatment groups are shownbelow:

    ______________________________________                                        Group Treatment    Dose     Adjuvant                                                                             Route  N                                   ______________________________________                                        I     None         None     None   None   11                                  II    HSV-1 gB + gD                                                                              25 μg +                                                                             Freund's                                                                             Footpad                                                                              11                                                     25 μg                                                   III   gP2          50 μg Freund's                                                                             Footpad                                                                              11                                  IV    Control,     None     Freund's                                                                             Footpad                                                                               9                                        Adjuvant only                                                           ______________________________________                                    

Animals were immunized on day 21 and again on day 42 by injection of thevaccines into the hind footpads. Both recombinant proteins gB1 and gD1were produced in mammalian cells as previously described. Results arereported in Table 3 and FIG. 15.

The results show that the pattern of recurrent herpetic disease was thesame for Groups I and IV, hence these groups were pooled for analysis(control, n=20).

                  TABLE 3                                                         ______________________________________                                                    Days Lesions                                                                             Recurrent                                                                              Percent                                                   Observed   Episodes Severe Re-                                                                            Days/                                 Group N     (Mean ± SE)                                                                           (Mean ± SE)                                                                         currences.sup.c                                                                       Episodes                              ______________________________________                                        Con-  20    15.9 ± 1.5                                                                            9.0 ± 0.7.sup.                                                                      19.4 ± 2.9.sup.                                                                    1.77                                  trol                                                                          gB +  11     9.0 ± 1.6.sup.b                                                                      6.6 ± 1.0.sup.a                                                                      7.1 ± 2.2.sup.b                                                                   1.36                                  gD                                                                            gP2   11    11.0 ± k0.9.sup.b                                                                     7.3 ± 0.7.sup.a                                                                     12.1 ± 3.1.sup.b                                                                   1.51                                  ______________________________________                                         .sup.a Glycoproteins (50 μg) administered with complete Freund's           adjuvant in the hind footpad 21 and again 42 days after intravaginal HSV2     challenge; recurrences scored day 21 through 92.                              .sup.b Significantly different from control (p < 0.05).                       .sup.d Percent recurrences with two or more herpetic lesions.            

Results shown in Table 3 and FIG. 15 indicate that vaccination with therecombinant glycoproteins has a significant impact on the frequence ofrecurrent disease. In addition, the gB+gD combination is better than themixture of natural glycoproteins.

The rate of recurrent disease as measured by the number of lesion daysoccurring within a specified time is an assessment that considers boththe frequency and the duration of recurrent episodes. FIG. 17A shows therate of recurrent herpetic infections, expressed as the mean number ofdays per week that herpetic lesions were noted. The immunized groupincludes both gBgD and gP-2 vaccinated animals. As shown in FIG. 17A.the rate of recurrent disease (lesion days per week) declined in allgroups as the period of evaluation became more remote to the initialinfection, but the rate of decline was greater in the vaccinatedanimals. The difference in the rates of recurrent herpetic infectionsbetween control animals and immunized animals is shown in FIG. 17B. Asseen in FIG. 17B, the effect of glycoprotein immunization on the rate ofrecurrent disease appeared to have been established following the firstimmunization dose rather than after the second dose, as might have beendeduced from FIG. 15.

6.3. Therapeutic Studies: Effect of recombinant HSV glycoproteinvaccines administered after primary infection on the host immuneresponse

The effect of post-infection glycoprotein administration on the hostimmune response was determined by measuring anti-HSV antibodies producedby the infected animals prior to infection, and after immunization withHSV glycoprotein vaccines.

The animals were inoculated with HSV-2 ms strain, treated withacyclovir, and treated with HSV glycoprotein vaccines as described inSection 6.2. Sera from the animals was collected on days 41 and 95.Anti-HSV antibodies in the sera was measured by ELISA, essentially asdescribed in Pachl, C., et al, J of Virology (1987) 61:315-325, which isthe procedure described in Section 1.6. The capture antigens includedHSV-1 glycoprotein mixture (gP-1), HSV-1 glycoprotein D (gD-1) or HSV-2glycoprotein D (gD-2).

The effects of HSV glycoprotein vaccine administration on anti-HSVantibody titers is shown in Table 4, where the data is expressed as thegeometric mean. Antibody was not detected in sera collected prior to HSVinoculation. As seen in Table 4, in the untreated control animalsanti-HSV antibody titers were greater on day 41 than on day 95. Incontrast, glycoprotein treated animals generally exhibited rising titersthrough day 95, and vaccination with HSV glycopoteins resulted insignificant increases in anti-HSV antibody titers (p<0.05) compared tothe untreated controls. Moreover, whereas treatment with the gP-2mixture produced a 1.4 to 7 fold increase in antibody titers, treatmentwith recombinant HSV-1 gBgD vaccine resulted in a 9 to 31 fold elevationin titers compared to control values. Thus, the administration of HSVglycoproteins to animals, and particularly recombinant HSV glycoproteinsgBgD, augments the host immune response and, as shown above in Section6.2, reduces the frequency and severity of recurrent HSV disease.

                  TABLE 4                                                         ______________________________________                                        Effects of HSV Glycoprotein Vaccine Administration                            after Recovery from Initial Genital Herpes on                                 Anti-HSV Antibody Titers in Guinea Pigs                                                           Anti-                                                                         HSV Antibody                                              Treat- gP-1 Antibody                                                                              gD-1 Antibody                                                                              gD-2 Antibody                                ment   Day 41  Day 95   Day 41 Day 95                                                                              Day 41                                                                              Day 95                             ______________________________________                                        Un-     548    474      32.5   14    65    48                                 treated                                                                       Adjuvant                                                                              818    754      22     34    25    68                                 only                                                                          gP-2   2796    3343     177    152   91    297                                gBgD   9891    14881    2444   4864  606   2391                               ______________________________________                                    

6.4. Therapeutic Studies: Effect of adjuvants on the immune responseinduced by HSV glycoprotein vaccines containing gD1

Several adjuvants were examined to determine their effect on theefficacy of immunotherapeutic treatment with HSV glycoprotein vaccines.The adjuvants tested were complete Freund's adjuvant (CFA), alum,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP).N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine referred to as nor-MDP),andN-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylaminereferred to as MTP-PE). In addition, an adjuvant (RIBl) containing threecomponents extracted from bacteria, monophosphoryl lipid A, trehalosedimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween80 emulsion was also tested.

The adjuvant effects were determined by measuring the amount of anti-gD1antibodies resulting from administration of gD1 containing vaccineswhich were also comprised of the various adjuvants. The vaccinescontaining gD1 alone serve as a model for gBgD containing vaccines,since the adjuvant effect is not expected to be specific to the type ofHSV glycoprotein in the vaccines.

In the following studies gD1 was synthesized in pHS132 and isolated asdescribed in Section 4.6. Female guinea pigs were given three footpadimmunizations consisting of 35 μg gD1 and various adjuvant formulationsat three week intervals. One week after the second immunization, andone, five, nine and thirteen weeks after the third immunization theanimals were bled and anti-gD titers were determined by ELISA. asdescribed in Section 1.6.

The results presented below are indicative that the most promising ofthe adjuvants tested are MTP-PE and RIBI formulations, since theyconsistently produce high anti-gD1 titers in experimental animals. Theselevels were equivalent to those seen with CFA, although the titers werenot maintained for as long a period of time as with CFA.

6.4.1. Comparison between CFA, alum, and thr-MDP

The animals were immunized with vaccines containing gD1 and either CFA,alum, thr-MDP, or thr-MDP plus alum. The effect of the adjuvants onanti-gD titers is shown in Table 5, where the data is expressed as thegeometric mean. As seen in Table 5, the most effective adjuvant was CFA.The highest antibody titers of the longest duration were seen in thegroup immunized with the CFA vaccine. The effect of the other adjuvantsis expressed as the percent of the titer obtained with CFA. The meantiters of those animals immunized with the thr-MDP adjuvant ranged fromapproximately 50 to 70% of the titers obtained with CFA. The lowestanti-gD1 titers were obtained using as adjuvant a 10% alum suspension,and the combination of the thr-MDP plus alum was less efficacious thanthr-MDP alone.

                                      TABLE 5                                     __________________________________________________________________________    HSV Adjuvant Study Mean ELISA Titers                                          Experiment A                                                                  Adjuvant  Number                                                                             Bleed 1                                                                              % of CFA                                                                            Bleed 2 % of CFA                                                                            Bleed 3 % of CFA                    __________________________________________________________________________    CFA/IFA   6(5) 13709 ± 2161                                                                      100   15066 ± 2878                                                                       100   16835 ± 5527                                                                       100                         Alum      4    1199 ± 467                                                                         9    1353 ± 318                                                                          9    1118 ± 211                                                                          7                          thr-MDP   6     9334 ± 2505                                                                      68    9641 ± 1696                                                                        64    7271 ± 1524                                                                        43                          thr-MDP + ALUM                                                                          6    2280 ± 501                                                                        17    5527 ± 1399                                                                        37    8333 ± 1707                                                                        50                          __________________________________________________________________________

6.4.2. Comparison of CFA, nor-MDP, and MTP-PE

The animals were immunized with vaccines containing gD1 and either CFA,nor-MDP, and MTP-PE. The MTP-PE was encapsulated in liposomes, and thislatter adjuvant was administered both with exogenous gD1, and with gD1incorporated into the liposomes. Liposomes were prepared by vortexingsynthetic phosphatidylcholine, phosphatidylserine and MTP-PE (or MTP-PE& gD1) at a ratio of 175:75:1 in suspension medium (sterile, isotonicDulbecco buffer PH 7.2, without Ca⁺⁺ and Mg⁺⁺ salts). As seen in Table6, immunization with vaccine containing CFA still yielded the highestanti-gD1 mean titer. The titers obtained with nor-MDP ranged from 44 to74% of the mean titers obtained with the group immunized with CFA. Themean titers obtained with MTP-PE and exogenous gD1 were somewhat lowerthan that obtained with nor-MDP, with a range from 32 to 72% of thoseobtained with CFA. The very low titers obtained with MTP-PE and liposomeencapsulated gD1 may be due to the very low levels of gD1 in theencapsulated form. The dose of encapsulated gD1 was only about 7% of theexogenous dose. This low dosage was due to the very low efficiency ofincorporation of gD1 into liposomes, which may have been caused by thesize of the antigen. Alternative formulations of liposomes could lead tomore efficient incorporation of the antigen.

                                      TABLE 6                                     __________________________________________________________________________    HSV Adjuvant Study Mean ELISA Titers                                          Experiment B                                                                  Adjuvant Number                                                                             Bleed 1                                                                              % of CFA                                                                            Bleed 2 % of CFA                                                                            Bleed 3 % of CFA                     __________________________________________________________________________    CFA/IFA  7(6) 8009 ± 1130                                                                       100   13962 ± 2304                                                                       100   8298 ± 896                                                                         100                          nor-MDP  7(6) 3519 ± 905                                                                        44    10387 ± 2946                                                                       74    5759 ± 920                                                                         69                           squalene/arlacel                                                              MTP-PE-  7(6) 4051 ± 891                                                                        51     9989 ± 1161                                                                       72    2653 ± 457                                                                         43                           Liposome                                                                      MTP-PE-gD-                                                                             7(6) 415 ± 218                                                                          5    1656 ± 175                                                                         12    n.d.    n.d.                         Liposome                                                                      __________________________________________________________________________

6.4.3. Comparison of CFA and RIBI

The animals were immunized with gD1 vaccines containing either CFA orRIBI. As seen in Table 7, the anti-gD1 mean titers of animals immunizedwith RIBI containing vaccine ranged from 60 to 104% of the titers ofanimals immunized with CFA containing vaccines. The highest anti-gD1titers obtained with RIBI were at bleed 2, where the titers surpassedthose obtained with CFA.

                                      TABLE 7                                     __________________________________________________________________________    HSV Adjuvant Study Mean ELISA Titers                                          Experiment C                                                                  Adjuvant                                                                             Number                                                                             Bleed 1                                                                              % of CFA                                                                            Bleed 2                                                                              % of CFA                                                                            Bleed 3                                                                              % of CFA                         __________________________________________________________________________    CFA/IFA                                                                              5(4) 7127 ± 5405                                                                       100   8315 ± 2604                                                                       100   6175 ± 1007                                                                       100                              RIBI   7(6) 5550 ± 2365                                                                        78   8638 ± 2566                                                                       104   3748 ± 1897                                                                        61                              __________________________________________________________________________

6.4.4. Comparison of RIBI, nor-MDP in low oil formulation, two componentRIBI, and two component RIBI plus nor-MDP

The high oil formulation of nor-MDP could be problematic due to sideeffects associated with immunization, such as redness and irritation atthe site of injections. These side effects could be overcome with loweroil formulation, and in addition, are more easily prepared and injected.

Low oil formulations of nor-MDP contained 4% Squalene and 0.0008% Tween80 as compared to the usual formulation of nor-MDP, which was 40%Squalene and 10% Aracel A.

Moreover, the cell wall skeleton component (CWS) in RIBI is a complex ofundefined character; therefore, it was desirable to substitute nor-MDPfor this component in RIBI (nor-RIBI) and to evaluate nor-RIBI withRIBI, and with two component RIBI which lacked CWS (RIBI-2).

The effect of these adjuvants on the anti-gD1 titer is shown in Table 8.As seen in the Table, CWS is an important component of RIBI, sinceomitting it caused significant decreases in the anti-gD1 titer at alltime points, and almost a 98% decrease in the titer at the early timepoint. However, the CWS component was effecitvely replaced by nor-MDP,since nor-RIBI was more efficacious as an adjuvant than was RIBI. Thelow oil formulation of nor-MDP was about 69% effective as RIBI at theearliest time point, but its effectiveness appeared to decrease withtime, and at the last time point, its effectiveness was about the sameas RIBI-2.

                                      TABLE 8                                     __________________________________________________________________________    HSV Adjuvant Study Mean ELISA Titers                                          Experiment D          % MPL +         % MPL +         % MPL +                 Adjuvant Number                                                                              Bleed 1                                                                              TDM + CWS                                                                              Bleed 2                                                                               TDM + CWS                                                                             Bleed 3                                                                              TDM                     __________________________________________________________________________                                                          + CWS                   RIBI     5(4)  4239 ± 1152                                                                       100      3205 ± 1460                                                                       100      1702 ± 405                                                                        100                     nor-RIBI 5     9859 ± 802                                                                        232      3292 ± 501                                                                        103      2351 ± 570                                                                        138                     nor-MDP, low oil                                                                       5(2)  2935 ± 1417                                                                        69      1822 ± 220                                                                         57       720 ± 130                                                                         42                     RIBI-2   4     72 ± 2                                                                             2       858 ± 619                                                                          27       743 ± 145                                                                         44                     __________________________________________________________________________

6.4.5. Comparison of RIBI, MTP-RIBI, and different formulationscontaining MTP-PE

The animals were immunized with gD1 containing vaccines formulated witheither RIBI, a derivative or RIBI in which the CWS component wassubstituted with MTP-PE (MTP-RIBI), MTP-PE in a high oil delivery system(Squalene/Arlacel). MTP-PE in a low oil delivery system. The low oilformulation of MTP-PE contained 4% Squalene and 0.0008% Tween 80. ThegD1 antibody titers obtained with these adjuvant formulations are shownin Table 9.

As seen in Table 9, the MTP-PE formulation was effective as an adjuvant,even when used as the only constituent in the low oil-detergentformulation. It was also an effective substitute for the CWS componentin RIBI, moreover, compared to RIBI its effectiveness increased withtime. At the third bleed, the titers obtained with MTP-RIBI were twicethat obtained with RIBI.

                                      TABLE 9                                     __________________________________________________________________________    HSV Adjuvant Study Mean ELISA Titers                                          Experiment E           % MPL +         % MPL +         % MPL +                Adjuvant Number                                                                              Bleed 1 TDM + CWS                                                                             Bleed 2 TDM + CWS                                                                             Bleed 3 TDM                    __________________________________________________________________________                                                           + CWS                  RIBI     5(4)  15912 ± 3191                                                                       100      8000 ± 1271                                                                       100     1318 ± 310                                                                         100                    MTP + RIBI                                                                             5      9218 ± 9411                                                                       58       9836 ± 1262                                                                       123     2678 ± 533                                                                         203                    MTP-PE   4     14803 ± 3751                                                                       93      14731 ± 3860                                                                       184     1181 ± 433                                                                          90                    squalene/arlacel                                                              MTP-PE, low oil                                                                        5     10694 ± 2135                                                                       67      17072 ± 3457                                                                       213     2455 ± 535                                                                         186                    __________________________________________________________________________

The MTP-PE adjuvant is simpler than RIBI, since MTP-PE contains only onecomponent and RIBI contains three components. Moreover, MTP-PE ispotentially safer than RIBI, since it is a defined chemical which issynthetic, and RIBI contains components which are isolated frombacteria. Therefore, MTP-PE may be a preferred adjuvant for theformulation of gBgD containing vaccines.

6.5. Therapeutic Studies: The effect of adjuvant, site ofadministration, and timing of administration on vaccine efficacy inpreventing subsequent recurrent herpetic disease in guinea pigs

The gBgD vaccine consisted of 25 μg each of recombinant gB1 and gD1purified to approximately 70-80% homogeneity as judged by SDS-PAGE. Therecombinant gB1 protein was a 50:50 mixture of gB1 prepared from cellline pHS113-9-10-21 and pHS137-7-B-50; these cell lines are CHO celllines which harbor the vectors pHS113 and pHS137, respectively. Thedescriptions for the preparation of pHS113 and pHS137 are in Section2.2. The gB protein was purified as described in Pachl et al. J ofVirology. 61:315-325 (1987), which is essentially as described inSection 6.1. The gD1 was prepared as described in Section 4.6, exceptthat during purification by affinity chromatography the anti-gD1monoclonal antibody C4D2 replaced 8D2.

The present example compares the efficacy of three adjuvants, nor-MDP,RIBI, and CFA. RIB1 consisted of a mixture of 50 μg of detoxifiedmonophorphoryl lipid A, 50 μg trehalose dimycolate, and 5 μg CSW perdose, presented in 2% squalene-Tween 80, all provided by RibiImmunochem. The adjuvant nor-MDP was used at 50 μg/dose emulsified with50% squalene/arlacel and the antigen.

The present study also compares two routes of administration, i.e..administration into footpads with administration which is intramuscularor subcutaneous. Finally, it compares various times of administration onthe prevention of subsequent recurrent herpetic disease in guinea pigs.The experimental design is shown in Table 10.

                  TABLE 10                                                        ______________________________________                                        Chrion 7 Experimental Design                                                          Treatment                                                             Group  N      Immunogen   Adjuvant                                                                              Route  Day.sup.c                            ______________________________________                                        1      11     none                                                            2      13     none.sup.a                                                      3      10     gBgD        CFA     FP     15,35                                4      12     gBgD        CFA     FP     21,42                                5      10     gBgD.sup.a  CFA     FP     21,42                                6      11     gBgD        none    IM/SC  21,42                                7      13     gBgD        Ribi    IM/SC  21,42                                8      10     gBgD        nor-MDP IM/SC  21,42                                .sup. 9.sup.b                                                                        --      --          --      --    --                                   10     10     gBgD        none    FP     21,42                                11     11     gBgD        Ribi    FP     21,42                                12     11     gBgD        nor-MDP FP     21,42                                13      6     gBgD        CFA     FP      8,28                                ______________________________________                                         .sup.a Daily vaginal swabs done d22-d100 to titer virus and assess            asymptomatic shedding.                                                        .sup.b Group 9 was eliminated.                                                .sup.c Day of administration of vaccine postinfection with initial virus      exposure on day 1.                                                       

Female Hartley guinea pigs weighing 350-400 g were intravaginallyinoculated with 5.7/log₁₀ pfu of HSV-2 strain MS on day 1. Animals wereconfirmed to be infected by recovery of HSV from vaginal swab samplescollected 24 hr after intravaginal inoculation. The clinical course ofinitial infection was monitored and quantitated by a gential skin lesionscore as described in Stanberry et al. J Infec Dis (1987) 155:914. Afterrecovery from initial infection animals were randomized for thetreatment groups shown in Table 10. Animals were examined daily forevidence of recurrent disease from days 11 to 100 after the resolutionof the acute disease. Lesion days are defined as days on which recurrentlesions are observed, severe recurrences are days when more than onevesicle is noted, and episodes are the occurrence of a new lesionfollowing a lesion free day.

The results obtained from analyses of the animals on days 22-76 arepresented in Table 11. The data in Table 11 suggests that for IMinjection, nor-MDP is more effective than RIB1 as an adjuvant; this isreflected in a lower total number of lesion days, a smaller percent ofsevere recurrences, and a diminishment in the total number of herpeticepisodes. Moreover, the vaccine containing nor-MDP and administered IMappeared to be as effective as the vaccine containing CFA andadministered in the footpads.

                  TABLE 11                                                        ______________________________________                                        Chiron 7                                                                      Effect of HSV-1 gBgD Vaccine Administered                                     After Intravaginal HSV-2 Inoculation                                          on Pattern of Recurrent Genital Herpes                                        Preliminary Analysis - Days 22-76                                                                    Total   Percent  Total                                 Group                  Lesion  Sever    Ep-                                   #     Treatment  N     Days*   Recurrences*                                                                           isodes*                               ______________________________________                                        1     Untreated  11    16.7 ± 2.1                                                                         29.0 ± 5.7                                                                          8.9 ± 0.8                          13    gBgD-Day 8-                                                                               5     9.0 ± 2.2                                                                         14.2 ± 3.9                                                                          5.8 ± 0.9                                CFA-FP                                                                  3     gBgD-Day 15-                                                                              9    10.0 ± 3.0                                                                         19.5 ± 7.4                                                                          6.2 ± 1.4                                CFA-FP                                                                  4     gBgD-Day 21-                                                                             11    11.6 ± 2.0                                                                         24.2 ± 3.2                                                                          7.3 ± 1.1                                CFA-FP                                                                  6     gBgD-Day 21-                                                                             10    14.2 ± 2.0                                                                         25.1 ± 5.3                                                                          8.0 ± 0.9                                No-Adj-IM                                                               7     gBgD-Day 21-                                                                             12    14.4 ± 2.2                                                                         29.8 ± 5.0                                                                          7.9 ± 0.9                                RIBI-IM                                                                 8     gBgD-Day 21-                                                                              8    10.0 ± 1.8                                                                         20.5 ± 4.3                                                                          6.4 ± 1.1                                nor-MDP-IM                                                              10    gBgD-Day 21-                                                                             10    14.5 ± 2.1                                                                         20.3 ± 3.1                                                                          8.9 ± 1.2                                No Adj-FP                                                               11    gBgD-Day 21-                                                                             11    10.7 ± 1.9                                                                         25.0 ± 4.2                                                                          6.9 ± 1.0                                RIBI-FP                                                                 12    gBgD-Day 21-                                                                              9    14.8 ± 1.9                                                                         25.4 ± 5.0                                                                          8.2 ± 0.7                                nor-MDP                                                                 ______________________________________                                         *Mean ± SE                                                            

The local reactions resulting from injection of the vaccines containingthe various adjuvants was also monitored, and the results are shown inTable 12. The incidence of local erythema and induration at the site ofinjection was the same for vaccines containing nor-MDP as for vaccinescontaining RIBI. Moreover, based upon the local reactogenicity, bothadjuvants appear to be acceptable for use in vaccines.

                  TABLE 12                                                        ______________________________________                                        Chiron 7 Reactions Summary                                                                         Eryth-                                                                              Indura-                                                                             Vac-  Ad-                                    Group N     ΔT°a                                                                      ema   tion  cine  juvant                                                                              Route                            ______________________________________                                        Vaccination #1                                                                .sup. 1.sup.d                                                                       11    ND.sup.b .sup. --.sup.c                                                                      --    O     O     O                                .sup. 4.sup.d                                                                       12    .08 ± .40                                                                           --    --    gBgD  CFA   FP                               6     12    .52 ± .27                                                                           4     0     gBgD  O     IM                               7     13    .20 ± .30                                                                           11    7     gBgD  RIBI  IM                               8     10    .00 ± .27                                                                           5     10    gBgD  nor-  IM                                                                      MDP                                    Vaccination #2                                                                1     11    .01 ± .46                                                                           --    --    O     O     O                                4     12    .98 ± .27                                                                           --    --    gBgD  CFA   FP                               6     12    .52 ± .27                                                                           4     O     gBgD  O     IM                               7     13    .27 ± .51                                                                           8     8     gBgD  RIBI  IM                               8      8    .01 ± .48                                                                           7     8     gBgD  nor-  IM                                                                      MDP                                    ______________________________________                                         .sup.a Temperature day 1 following vaccine  day 0 for vaccine (mean ±      SD) (i.e., D22-21 or D 43-42)                                                 .sup.b ND  not done                                                           .sup.c Not applicable                                                         .sup.d All animals had R let naired prior to vaccination except these    

The results in Table 11 also show that the relative efficacy oftreatment increases as the interval between the initiation ofimmunotherapy and the onset of acute disease decreases. In animals whichhad received gBgD vaccine containing CFA beginning 8, 15, or 21 daysafter the initial infection, those animals which had received thevaccine the shortest time after intravaginal inoculation suffered thesmallest number of lesion days, had the lowest percent of severerecurrences, and the fewest total episodes compared to the untreatedcontrol. The values obtained for the animals vaccinated 15 days afterinfection was higher than those obtained for the 8 day vaccinationgroup, and the 21 day group was higher than the 15 day group. Thiseffect is also shown in FIG. 18, which presents a graph of the number ofrecurrences on the days after intravaginal inoculation for animals whichwere initially vaccinated 8, 15, or 21 days after the HSV-2 inocluation.

The data in FIG. 18 was used to calculate the percent reduction in therates of recurrent disease (See Section 6.2 for an explanation of thesignificance of the rate of recurrent disease). This data is presentedin Table 13. where it may be seen that at earliest time periods, i.e..14-50 days, the greatest percent reduction in the rate of recurrentdisease was obtained by giving the initial vaccination at 8 days.However, from 51-92 days, the most effective protection was obtained bygiving the initial vaccination 15 days after the intravaginalinoculation with HSV. The least protection occurred when the initialvaccination was given 21 days after the initial exposure to HSV-2.

                  TABLE 13                                                        ______________________________________                                        Chiron 7                                                                      (Weekly Rate) Rates of Recurrence After Glycoprotein Treatment                ______________________________________                                        Day 8       14-29   30-50     51-71 72-92                                     ______________________________________                                        UNRx        3.30    2.48      1.42  1.15                                      gBgD        1.75    0.80      1.0   0.94                                      % Control   53.0%   32.3%     70.4  81.7                                      % Reduction 47.0%   67.7%     29.6% 18.3%                                     ______________________________________                                        Day 15      16-36   37-57     58-78 79-85                                     ______________________________________                                        UNRx        3.27    2.09      1.42  0.97                                      gBgD        2.03    1.04      0.81  0.64                                      % Control   62.0%   49.8%     57.0% 66.0%                                     % Reduction 38.0%   50.2%     43.0% 34.0%                                     ______________________________________                                        Day 21      22-42   43-63     64-84 85-92                                     ______________________________________                                        UNRx        2.82    2.18      1.03  0.85                                      gBgD        2.09    1.46      0.76  1.33                                      % Control   74.1%   67.0%     73.8% 156.5%                                    % Reduction 25.9%   33.0%     26.2% 56.5%                                     ______________________________________                                    

According to the present invention, novel vaccines are providedeffective against Herpes Simplex Virus Types 1 and 2 administered eitherpre-viral infection or post-viral infection.

Although the foregoing invention has been described in some detail byway of illustration and example for purpose of clarity andunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. A vaccine formulation consisting essentially ofherpes simplex virus (HSV) polypeptides wherein the HSV polypeptidesare:(a) immunogenic; (b) glycosylated; and (c) consist of:(i) a HSVglycoprotein B polypeptide or immunogenic fragments thereof; and (ii) aHSV glycoprotein D polypeptide or immunogenic fragments thereof.
 2. Avaccine according to claim 1, wherein the glycoprotein B is a Type 1glycoprotein B and the glycoprotein D is a Type 1 glycoprotein D.
 3. Avaccine according to claim 1, wherein the glycoprotein B is a Type 1glycoprotein B and the glycoprotein D is a Type 2 glycoprotein D.
 4. Avaccine according to claim 1, wherein the glycoprotein B is a Type 2glycoprotein B and the glycoprotein D is a Type 1 glycoprotein D.
 5. Avaccine according to claim 1, wherein the glycoprotein B is a Type 2glycoprotein B and the glycoprotein D is a Type 2 glycoprotein D.
 6. Avaccine according to claim 1, wherein the glycoprotein B is a Type 1 anda Type 2 glycoprotein B and the glycoprotein D is a Type 1 and a Type 2glycoprotein D.
 7. A vaccine according to claim 1, wherein thepolypeptides include an anchor sequence.
 8. A vaccine according to claim1, wherein the polypeptides are substantially free of an anchorsequence.
 9. A vaccine according to claim 1, wherein the polypeptidesare each of molecular weight about 1500 to 5000 daltons.
 10. A vaccineaccording to claim 1, wherein said vaccine includes a pharmacologicallyacceptable carrier.
 11. A vaccine according to claim 1, wherein saidvaccine includes an adjuvant.
 12. A method for immunizing a mammalagainst herpes simplex virus infection comprising vaccinating the mammalwith the vaccine of claim 1, wherein the mammal is vaccinated subsequentto a primary infection with herpes simplex virus.
 13. A method ofreducing the rate of recurrent herpetic disease in a mammal that hasbeen subjected to primary infection with herpes simplex virus comprisingadministering the vaccine of claim 1 to the mammal.
 14. A vaccineaccording to claim 1, wherein the HSV glycoprotein B polypeptide issubstantially free of an anchor sequence.
 15. A vaccine according toclaim 11, wherein the adjuvant is alum.
 16. A vaccine according to claim11, wherein the adjuvant isN-acetyl-nor-muramyl-L-alanyl-D-isoglutamine.
 17. A vaccine according toclaim 11, wherein the adjuvant is formulated in a low oil formulation.18. A vaccine according to claim 11, wherein the adjuvant is 4% Squaleneand 0.0008% Tween
 80. 19. A vaccine according to claim 11, wherein theadjuvant isN-acetylmuramyl-L-analyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine.20. A vaccine according to claim 19, wherein the adjuvant is formulatedin a low oil formulation.
 21. A vaccine according to claim 20, whereinthe adjuvant is formulated in 4% Squalene and 0.0008% Tween
 80. 22. Avaccine according to claim 11, wherein at least one of theimmunologically active polypeptides and the adjuvant are encapsulated ina liposome.
 23. A vaccine according to claim 22, wherein the adjuvant isN-acetylmuramyl-L-analyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine.